- Email: [email protected]
How Does Streptococcus pneumoniae Invade the Brain?
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Review
How Does Streptococcus pneumoniae Invade the Brain? Federico Iovino,1,2,* Jolien Seinen,3 Birgitta Henriques-Normark,1,2,4 and Jan Maarten van[15_TD$IF] Dijl3,4 Streptococcus pneumoniae (the pneumococcus) is the major cause of bacterial meningitis. The mechanisms by which pneumococci from the bloodstream penetrate the blood–brain barrier to reach the brain are not fully understood. Receptor-mediated adhesion of the bacteria to the brain endothelium is considered a key event leading to meningitis development. The aim of this review is to discuss recent advances and perspectives related to the interactions of S. pneumoniae with the blood–brain barrier during the events leading to meningitis. Altogether, the available data suggest that, by precisely defining the pathways and ligands by which S. pneumoniae adheres to specific receptors, it may be possible to interfere with the respective mechanisms and develop strategies to prevent or even cure pneumococcal meningitis. Streptococcus pneumoniae, the Blood–Brain Barrier, and Pneumococcal Meningitis The Gram-positive bacterium [26_TD$IF]Streptococcus pneumoniae (the pneumococcus) is an important commensal resident of the human nasopharynx. Although carriage is usually asymptomatic, S. pneumoniae can become invasive and spread from the upper respiratory tract to other organs, leading to serious diseases such as pneumonia, sepsis, or meningitis [1,2]. S. pneumoniae is the major etiological cause of bacterial meningitis, responsible for two-thirds of meningitis cases in Europe and in the USA [3–5]. Bacterial meningitis is a disease with high morbidity and mortality worldwide despite the implementation of several vaccination programs and antimicrobial agents [3–6]. A major route for bacteria to reach the meninges is through the bloodstream [7]. Having reached the blood vessels in the brain, bacteria present in the bloodstream have to cross the blood–brain barrier to enter the brain and cause infection. This view is supported by recent immunofluorescent analyses combined with high-resolution confocal microscopy, where blood-borne S. pneumoniae was clearly shown to adhere to the brain vascular endothelium prior to invasion of the brain [8]. The blood–brain barrier is a specialized vasculature system that separates the brain from circulating blood and has critical functions in both protection and nutrient supply of the brain [9–11]. Endothelial cells form the layer that lines the interior surface of the blood vessels [12,13]. Pathogens can invade the brain only after crossing the endothelial cell layer of the blood–brain barrier and, therefore, they must develop strategies to pass this barrier. The inflammatory features and clinical complications following bacterial meningitis normally observed in humans can be reproduced using animal models. Notably, most in vivo models that examine the pathophysiology of bacterial meningitis involve the direct injection of pneumococci into the brain of mice or rats [14–18]. Administration of bacteria directly into the brain bypasses the need for blood–brain barrier translocation. In order to study the interaction of blood-borne
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Trends Streptococcus pneumoniae, the main causative agent of bacterial meningitis, penetrates the blood–brain barrier by binding to PECAM-1 and pIgR expressed by brain vascular endothelial cells. Overall, recent findings suggest that PECAM-1 and pIgR, together with platelet-activating factor receptor (PAFR), another receptor thought to be more involved in signaling rather than physical binding, might cooperate during adhesion of pneumococci to brain endothelial cells. The passage of S. pneumoniae across the blood–brain barrier is a crucial step for meningitis development. The knowledge of how S. pneumoniae penetrates the blood–brain barrier can be used to develop strategies aimed at interfering with the respective pathways in order to prevent the entry of the pathogen into the brain.
1 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE 171 77, Stockholm, Sweden 2 Department of Clinical Microbiology, Karolinska University Hospital, SE 171 76, Stockholm, Sweden 3 Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 4 These authors contributed equally to this work.
*Correspondence: [email protected] (F. Iovino).
http://dx.doi.org/10.1016/j.tim.2015.12.012 © 2016 Elsevier Ltd. All rights reserved.
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S. pneumoniae with the brain vascular endothelium in vivo, prior to the onset of meningitis, a socalled bacteremia-derived meningitis model was established in recent years [8,19]. This model, where bacteria are injected into the bloodstream instead of the brain, has allowed the visualization of successive steps from bacteremia to brain invasion. The aim of the present review is to discuss recent advances in our understanding how blood-borne S. pneumoniae interacts with the blood–brain barrier endothelium during the events leading up to meningitis, and how to use such knowledge to develop new therapeutic strategies to fight the disease.
Receptor-Mediated Transcytosis How bacterial pathogens cross the blood–brain barrier is still subject [27_TD$IF]of debate. Several possible mechanisms have been implicated in this process, such as: (i) destruction of the endothelial cell layers in case of, for example, Neisseria meningitidis [20]; (ii) traversal of the blood–brain barrier in between the endothelial cells by disruption of tight junctions in case of group A streptococci [21]; and (iii) receptor-mediated transcytosis across endothelial and epithelial cell layers in case of S. pneumoniae [22,23]. The view that pneumococci pass the blood–brain barrier via transcytosis rather than pericellularly is supported by experiments, showing a continuous and homogeneous staining of VE-Cadherin in the brain of infected mice [8]. This implies that blood-borne S. pneumoniae does not cause a disruption of the endothelial tight junctions. In this context, it should be noted that bacterial pathogens such as pneumococci have the capability to bind to certain receptors on the plasma membrane of epithelial and endothelial cells, and this receptormediated binding facilitates bacterial invasion into[3_TD$IF] and translocation over[3_TD$IF] human cell layers. The passage of bacterial pathogens across such layers is a fundamental step for development of invasive diseases and it is necessary for S. pneumoniae to cross the blood–brain barrier to develop bacterial meningitis [22,23]. The Platelet-Activating Factor Receptor (PAFR) The first reported receptor implicated in adhesion to, invasion of, and also transcytosis through[3_TD$IF] endothelial cells is the [28_TD$IF]PAFR[6_TD$IF]. PAFR is a G-protein-coupled receptor with seven transmembrane domains and its natural ligand is the platelet-activating factor (PAF). PAF is a mediator in diverse pathologic processes, such as allergy, asthma, septic shock, arterial thrombosis, and inflammation [24–26]. Binding of PAF to the PAFR stimulates numerous signal transduction pathways including phospholipase C, D, A2, mitogen-activated protein kinases (MAPKs), and the phosphatidylinositol-calcium second messenger system [25]. PAFR has been proposed to bind S. pneumoniae, thereby facilitating adhesion to, uptake by, and transcytosis through[3_TD$IF] endothelial cells leading to invasive disease [23,27,28]. However, while in vitro and in vivo studies indicate that PAFR is involved in the development of invasive pneumococcal disease (IPD), there is so far no unequivocal published evidence that a direct binding between S. pneumoniae and PAFR occurs [24]. A recent study suggests that PAFR plays a role in pneumococcal adhesion to endothelial cells, even though pneumococci do not directly bind to PAFR [29]. After immunofluorescent microscopy analysis, colocalization of PAFR and S. pneumoniae was not observed in vivo in mouse brain tissue nor in endothelial cells in vitro. Nevertheless, upon blockade of PAFR, adhesion of pneumococci to human endothelial cells was significantly reduced, indicating that PAFR most likely has an indirect role in IPD [29]. When the PAFR was genetically deleted or chemically inhibited, S. pneumoniae was still able to adhere to human cells in vitro, and to cause invasive disease in vivo [23,27]. The Laminin Receptor (LR) The [29_TD$IF]LR[6_TD$IF] is an important molecule involved both in cell adhesion to the basement membrane and in signal transduction following this binding event. The LR on endothelial cells was found to interact with neurotropic viruses, including Sindbis virus [30], Dengue virus [31], adenoassociated virus [32], tick-borne encephalitis virus, and Venezuelan equine encephalitis virus [33]. LR was identified to be a common receptor for both S. pneumoniae and N. meningitidis on
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the surface of rodent and human brain microvascular endothelial cells [19]. Fluorescent beads coated with the pneumococcal choline-binding protein (Cbp) A [34–36] that were injected intravascularly into mice adhered to the cerebral endothelium. Pretreatment with anti-LR antibody led to an inhibition of the adherence of the CbpA–beads to the endothelium in mice, thus suggesting that the LR interacts with the bacteria[3_TD$IF] facilitating translocation of intracellular pneumococci across the blood–brain barrier endothelium, from the basolateral side of endothelial cells to the brain tissue [19]. The Poly Immunoglobulin Receptor (pIgR) The [30_TD$IF]pIgR[6_TD$IF] transports immunoglobulins across mucosal epithelial barriers (e.g., in the respiratory tract) as a first line of defense to antigens from pathogenic bacteria [37–39]. S. pneumoniae has been shown to bind to pIgR expressed by human nasopharyngeal epithelial cells[3_TD$IF] and this binding facilitates pneumococcal translocation through the nasopharyngeal epithelium [22]. When human nasopharyngeal epithelial (Detroit) cells were treated with an antibody against pIgR prior to pneumococcal challenge, adherence was reduced [22]. This implies that pIgR is functionally involved in pneumococcal adhesion to nasopharyngeal epithelium. Furthermore, immunoblotting analyses showed that pneumococcal CbpA binds to human pIgR, indicating that CbpA may be required to mediate binding of S. pneumoniae to this receptor [22]. PIgR is not only present on the epithelium of the respiratory tract, but can also be found on the plasma membrane of endothelial cells, especially brain endothelial cells [29]. Due to its presence on the blood–brain barrier endothelium, the possible involvement of pIgR in pneumococcal adhesion to brain endothelial cells was recently investigated [29]. Notably, immunofluorescent analyses of brain tissue of infected mice showed that most pneumococci adhering to the brain vasculature were colocalized with pIgR [29]. Furthermore, by incubating pneumococci with human endothelial cell lysates, it was shown that S. pneumoniae can directly bind to pIgR [29]. The combined results therefore suggest that pIgR is a direct adhesion receptor for S. pneumoniae on the blood–brain barrier endothelium. Even though the main route of brain invasion seems to be the bloodstream, pneumococci may also reach the brain through skull fractures that tear the dura mater, or directly from the nasopharynx. In fact, it was recently reported that S. pneumoniae can transmigrate from the nasopharyngeal epithelium to the olfactory nerves that anatomically connect the upper respiratory tract with the central nervous system [40]. Although it was not shown experimentally, an intriguing hypothesis is that the pIgR on the nasopharyngeal epithelium could mediate the passage of the bacteria from the epithelial cells to the olfactory nerve. Clearly, once this passage has occurred the bacteria can travel along the nerve and reach the brain. Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) The [31_TD$IF]PECAM-1 ([10_TD$IF]also known as CD31) is a pan-endothelial protein[3_TD$IF] present in the intercellular junctions of the endothelial cells, that is involved in leukocyte migration, angiogenesis, and integrin activation [41–43]. In particular, the involvement of PECAM-1 in leukocyte–endothelium interaction and leukocyte trans-endothelial migration in the vascular system makes PECAM-1 a key molecule in inflammation and neuroinflammation [43]. Recently, PECAM-1 was implicated in gastrointestinal infection caused by Salmonella enterica serovar Typhimurium [44], suggesting an active role of PECAM-1 in host–pathogen interactions. This raised the question whether PECAM-1 might also play a role in host–pneumococcal interactions. Indeed, for the first time it was recently shown that, in brain tissue from intravenously infected mice, most pneumococci did colocalize with PECAM-1 [45]. Blockade of PECAM-1 with a specific antibody in human brain endothelial cells led to a significant reduction of bacterial adhesion, indicating that PECAM-1 is functionally involved in S. pneumoniae adhesion to the brain vascular endothelium [45]. Using a biochemical approach, it was demonstrated that S. pneumoniae can directly bind PECAM-1 expressed by human endothelial cells [45]. Importantly, PECAM-1 colocalized with pIgR on the
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Key Figure
Cooperation of Platelet-Activating Factor Receptor (PAFR), pIgR, and PECAM-1 in Pneumococcal Adhesion to Endothelial Cells: A Proposed New Model (A)
(B) S. pneumoniae
Inflammation
Inflammatory cytokines
Pneumococcal PAF components
plgR
S. pneumoniae binding sites
PAFR before acvaon
More S. pneumoniae binding sites
More adhering pneumococci
PAFR activation PAFR
plgR
Blood
plgR-PECAM-1 upregulation
Endothelial cell
Brain
Figure 1. (A) Under normal physiological conditions, PAFR is present on the plasma membrane of endothelial cells in its non-activated state, while pIgR and PECAM-1 are forming a double receptor displaying binding sites that can potentially be used as anchors for the adhesion of bacterial pathogens.[1_TD$IF] (B) Upon pneumococcal infection, inflammation develops and PAFR is activated by pneumococcal components, such as pneumolysin and choline-binding proteins, and PAF. Activation of PAFR may lead to the upregulation of pIgR–PECAM-1. This enhanced expression of pIgR–PECAM-1 would make more potential binding sites available for Streptococcus pneumoniae and, ultimately, this would lead to more pneumococci adhering to endothelial cells, allowing them to enter the brain.
brain endothelium and, moreover, the two receptors directly interacted with each other as demonstrated by co-immunoprecipitation experiments suggesting the possible formation of a double receptor that facilitates the passage of S. pneumoniae across the blood–brain barrier [45] (Figure 1, Key Figure). Nevertheless, to unequivocally demonstrate the joint role of PECAM-1 and pIgR in pneumococcal meningitis, S. pneumoniae should be intravenously administered to PECAM-1 / [2_TD$IF] and pIgR / mice. In the case of a direct role, as suspected, one would expect that only few bacteria can translocate the blood–brain barrier in the knock-out mice. Although current literature still lacks such experimental evidence, it was previously shown that adhesion of bacteria to the brain vasculature endothelium represents a crucial moment for invasion of meningeal pathogens in the brain. In addition, the observation that blood-borne pneumococci colocalize with both PECAM-1 and pIgR in vivo strongly indicates that the two receptors jointly facilitate the passage of the bacteria from the bloodstream into the brain [29,45].
Cooperation of PAFR, pIgR and PECAM-1 during Pneumococcal Adhesion to Endothelial Cells: A New Model From the current literature [24], it remains unclear what the exact role of PAFR is in IPD. The involvement of PAFR in S. pneumoniae adhesion to endothelial cells might well be exerted through the binding of PAF or pneumococcal cell-wall components to the receptor and the subsequent inflammatory consequences of its activation [24]. Inflammation leads to an upregulation of receptors expressed on the plasma membrane of the apical side of the cells [7,27,46], and PECAM-1 and pIgR may be among them. In the presence of more pIgR and PECAM-1,
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S. pneumoniae has more sites to adhere to on the endothelial cells. It has been repeatedly shown that blocking PAFR with a specific antibody or antagonist leads to a significant reduction of pneumococcal adhesion to endothelial cells in vitro [23,27,29]. We therefore hypothesize that, due to PAFR blocking, the upregulation of pIgR and PECAM-1 may be prohibited. Possibly, the presence of pneumococci may trigger the autocrine production of PAF, directly or indirectly, which activates PAFR, leading to an upregulation of pIgR and PECAM-1. This would increase pneumococcal adherence to pIgR and PECAM-1 and, in turn, more adherent bacteria would increase the chances of bacterial invasion into the cells (Figure 1). Whether PAFR activation leads, directly or indirectly, to pIgR-PECAM-1 upregulation still remains an open question. Besides PAF, secreted pneumococcal components, such as pneumolysin [47,48] and CbpA, could bind to PAFR and activate this receptor [49]. Clearly, this idea still needs to be experimentally validated. Accordingly, an in-depth investigation of secreted and surface-attached pneumococcal proteins, including proteomics and structural analyses, will be required to identify any protein(s) of S. pneumoniae that could be recognized and bound by PAFR. Interesting candidate proteins for such studies were recently identified by Pribyl et al. in a proteomics-based survey of the pneumococcal surface-bound and secreted proteins [50]. An important recent finding was that if pIgR and PECAM-1 are blocked simultaneously, pneumococcal adhesion to endothelial cells is reduced even more than when the two receptors are blocked individually [45]. Bacteria cannot adhere to pIgR and PECAM-1 after blocking with their respective antibodies, but PAFR activation may still be possible and could thus lead to upregulation of PECAM-1 and pIgR expression.
Humans Deficient in Receptors PAFR, pIgR, and PECAM-1 Have Not Been Identified, but Mice Survive without Them Humans deficient in PAFR have not been identified. However PAFR–/– mice have been generated [51]. In PAFR–/– mice, modifications of known angiogenic factors, such as vascular endothelial growth factor (VEGF), have not been observed, but a sustained rise in the chemokines CXCL2 and CCL2 has been detected, which may compensate for the absence of PAFR as an important molecule in maintaining immunological surveillance [52]. These findings indicate that the absence of PAFR in mice is compatible with health, although it has to be noted that this was observed under controlled conditions. Mice lacking pIgR were of normal size and fertility as well, but displayed increased IgG levels in their sera, suggesting a triggering of systemic immunity [53]. These findings indicate that absence of pIgR is not lethal either, but at the same time it seems that absence of pIgR is sensed as a potential danger by the systemic immune system [38,53]. PECAM-1 is expressed not only by endothelial cells but also by platelets, neutrophils, and macrophages, and its signaling pathways might mediate resistance to apoptosis and promote cell survival [41,42]. Absence of PECAM-1 may therefore lead to several cellular dysfunctions. Interestingly, PECAM-1 was reported not to be required for embryogenesis, fetal maturation, or fertility in mice, and, furthermore, PECAM-1 expression on platelets is not essential for platelet–platelet aggregation [54]. PECAM-1–/– animals develop normally and do not show any signs of an abnormal physiology under normal baseline conditions. However, as the animals age they can develop an autoimmune lupus-like syndrome and, when stressed, a variety of abnormal responses have also been noticed. In particular, PECAM-1–/– animals exhibited a prolonged bleeding time [55]. Thus, although the absence of the PAFR, pIgR, or PECAM-1 receptors did not cause serious problems in mice during certain circumstances, dysfunctions and abnormalities due to a lack of these receptors cannot be excluded.
Blockade of S. pneumoniae Receptors: A New Avenue for the Prevention and Cure of Pneumococcal Meningitis Knowledge of the mechanisms by which the pneumococcus interacts with the blood–brain barrier before invading the brain is fundamental in order to develop therapeutic strategies to avoid adhesion of S. pneumoniae to the blood–brain barrier and, thus, invasion of the pathogen
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into the central nervous system. Adhesion of pneumococci to the blood–brain barrier is spatiotemporally controlled at different sites throughout the brain, and the local immune system in the brain is activated immediately as soon as bacteria in the bloodstream interact with the blood–brain barrier [8]. The bacteremia-derived meningitis model is used to investigate the interactions of bacteria with the blood–brain barrier, including the events preceding meningitis [8,19,29,45]. To study meningitis itself, intracisternal administration of bacteria was previously used to induce meningitis in vivo [14–18]. Using this model, neutrophil influx into the brain was detected only at 30 hours after challenge, indicating that white blood cells can be detected in the brain only at late stages of the disease [14]. A high count of white blood cells in the cerebrospinal fluid (CSF) is often used as a clinical test for diagnosis of meningitis [56]. Moreover, classic symptoms of meningitis, such as rash, neck stiffness, photophobia, severe headache, and impaired consciousness[3_TD$IF] develop late [56–58]. While these symptoms represent the classical picture of suspected meningitis, the disease may be already at an advanced stage in patients displaying them. Thus, it is not really surprising that, at this stage, the risks of brain damage are considerable even if the infection is successfully fought with antibiotics. World Health Organization (WHO) data indicate that, even if the disease is diagnosed early and adequate treatment is started immediately, 5–10% of the patients will die, typically within 24[32_TD$IF]–48 hours after occurrence of the first symptoms. When left untreated, up to 50% of the patients may die. In case of recovery, brain damage, hearing loss, or a learning disability appears in 10–20% of the survivors (http://www.who.int/topics/meningitis/en/). It is therefore crucial to start interventions before bacteria have invaded the brain in order to optimize the survival chances of patients and to prevent permanent damage. Treatment with antibiotics is the clinically used way to cure meningitis, and routine vaccination against pneumococcal infections with the pneumococcal conjugate vaccine (PCV), which is active against seven to thirteen common capsular serotypes of this pathogen, has significantly reduced the incidence of invasive pneumococcal disease in vaccinated children [59–61]. Knowing that PAFR, pIgR, and PECAM-1 mediate the first adhesion of blood-borne S. pneumoniae to the vascular endothelium of the blood–brain barrier opens a complementary approach to antibiotics and vaccines for curing or preventing pneumococcal meningitis by developing therapies that interfere with these receptors. This could, for example, be achieved by blocking PAFR, pIgR, and PECAM-1 with specific antibodies which would limit the adhesion of pneumococci to the blood–brain barrier and then observing whether the systemic administration of blocking antibodies leads to less severe disease symptoms. Alternatively, blockade of the receptors can be achieved by using either chemical antagonists, such as, for instance, L659989 or WEB2086 previously used to successfully block the PAFR in vitro [24], or the pneumococcal proteins that should specifically anchor to the receptors, such as CbpA that was reported to bind to pIgR in the respiratory epithelium [22,62]. Another alternative, but technically more challenging approach, is to prevent binding of S. pneumoniae to PAFR, pIgR, and PECAM-1 on the blood–brain barrier endothelium by blocking the respective ligands on the pneumococcal cell surface with engineered soluble derivatives of these receptors. This would have the potential advantage of minimal interference with the physiological functions of these three receptors. Such studies could be aided by targeted infection-imaging approaches, for instance, with positron emission tomography (PET) tracers that bind well to S. pneumoniae [63]. However, while blocking of these pneumococcal receptors could provide protection against meningitis, it will be crucial to develop strategies to avoid possible dysfunctions caused by their blocking before this approach can be implemented in the clinic. Based on literature data, mutated PAFR in mice is not likely to cause serious dysfunctions [51]. However, mutated pIgR may interfere with the normal transport of immunoglobulins across cells. It has been shown that treatment with phorbol myristyl acetate (PMA) activates isozymes of protein kinase C (PKC), a downstream protein in the signaling cascade of pIgR [[3_TD$IF]64]. This activation stimulates not only pIgR transcytosis, but more generally the pathway of a variety of molecules in the transcytotic pathways [[3_TD$IF]64]. Thus, treatment with PMA can stimulate cells to use other transcytotic systems and their receptors, in case immunoglobulin transfer mediated by pIgR is altered. In this way
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transport of immunoglobulins can be maintained through other transcytotic pathways. Regarding the vascular role of PECAM-1, it is important to bear in mind that endothelial cells express a vast range of endothelial adhesion factors (such as VECAM-1 and ICAM-1) that perform similar functions as PECAM-1 [[34_TD$IF]65,66]. Therefore, is conceivable that such adhesion molecules could complement for PECAM-1's role in case PECAM-1 is blocked. In principle, it should thus be possible to develop monoclonal antibodies blocking the pneumococcal binding site of the PECAM-1 receptor. While the blocking of pIgR and PECAM-1 showed only a partial reduction of pneumococcal adherence to endothelial cells [45], the blocking of the PAFR, pIgR, and PECAM-1 receptors at the same time could be a potential way to completely preclude the adhesion of S. pneumoniae to the blood–brain barrier endothelium and, therefore, prevent completely pneumococcal invasion into the brain. This, however, remains to be assessed experimentally. Clearly, an important advantage of conceivable therapies based on monoclonal antibodies against PAFR, pIgR, and PECAM-1 would be that these antibodies need to be administered only as long as pneumococci are detectable in the blood. Once these have been fought successfully with regular antibiotics, the antibody therapy can be stopped, which will limit possible unwanted side effects to a minimum.
Outstanding Questions How exactly does the signaling receptor PAFR cooperate with the binding receptors pIgR and PECAM-1 during the adhesion of S. pneumoniae to brain endothelial cells? Does an absence of adhesion receptors lead to less bacterial invasion into the brain in animal meningitis models? Is the treatment with blocking antibodies/antagonists/receptor ligands a valid way to block the adhesion receptors and prevent bacterial passage across the blood–brain barrier? If so, can such treatment be applied in humans?
Lastly, it has been demonstrated that pneumococci in the blood can reach and adhere to the blood–brain barrier endothelium after which they may invade the brain [8]. Bacteremia is present in 20–30% of cases with pneumococcal pneumonia, which can occur when an infection of the lungs grows out of control [[35_TD$IF]67–[36_TD$IF]69]. Patients with bacteremia and pneumonia can be considered ‘high-risk’ for progression towards brain infection and, for this reason, it could be valuable for them to receive innovative treatments that block pneumococcal receptors. This would prevent pneumococci from adhering to the blood–brain barrier and invading the brain. Such a treatment would even be of relevance for patients who are hospitalized with meningitis as it would still reduce the numbers of bacteria adhering to the blood–brain barrier and, in combination with antibiotic therapy, both the blood-borne bacteria and the bacteria that have already reached the brain could be eliminated.
Concluding Remarks What is the ultimate goal of knowing how S. pneumoniae invades the brain? Clearly, if pneumococci cannot adhere to endothelial cells, bacterial invasion will most likely be prevented. Therefore, by precisely defining the pathways by which S. pneumoniae adheres to specific receptors, it may be possible to develop drugs that interfere with these mechanisms and to develop strategies to prevent or even cure pneumococcal meningitis, as this still remains a very serious disease with high mortality and morbidity worldwide[37_TD$IF] (see Outstanding Questions). Judged by our current understanding of pneumococcal brain invasion, this is a highly ambitious but potentially achievable objective. Acknowledgments We thank Staffan Normark, Jetta Bijlsma and Ingrid Molema for their critical feedback[38_TD$IF]. F.I. and B.H.-N. were supported by grants from the Swedish Research Council, ALF grant from Stockholm County Council, the Foundation for Strategic Research (SSF), and Knut and Alice Wallenberg foundation. F.I. and J.S. were supported in part by grants from the Graduate School of Medical Sciences of the University of Groningen. J.S. and J.M.v.D. acknowledge the Deutsche Forschungsgemeinschaft Research Training Group GRK1870 for support.
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40. van Ginkel, F.W. et al. (2004) Pneumococcal carriage results in ganglioside-mediated olfactory tissue infection. Proc. Natl. Acad. Sci. U.S.A. 100, 14363–14367 41. Privratsky, J.R. and Newman, P.J. (2014) PECAM-1: regulator of endothelial junctional integrity. Cell Tissue Res. 355, 607–619 42. Newman, P.J. and Newman, D.K. (2003) Signal transduction pathways mediated by PECAM-1: new roles for an old molecule in platelet and vascular cell biology. Arterioscler. Thromb. Vasc. Biol. 23, 953–964 43. Kalinowska, A. and Losy, J. (2006) PECAM-1, a key player in neuroinflammation. Eur. J. Neurol. 13, 1284–1290 44. Lovelace, M.D. et al. (2013) Absence of platelet endothelial cell adhesion molecule 1, PECAM-1/CD31, In Vivo increases resistance to Salmonella enterica serovar Typhimurium in mice. Infect. Immun. 81, 1952–1963 45. Iovino, F. et al. (2014) Platelet endothelial cell adhesion molecule-1, a putative receptor for the adhesion of Streptococcus pneumoniae to the vascular endothelium of the blood–brain barrier. Infect. Immun. 82, 3555–3566 46. Shukla, S.D. et al. (2014) Airway epithelial platelet-activating factor receptor expression is markedly upregulated in chronic obstructive pulmonary disease. Int. J. Chron. Obstruct. Pulmon. Dis. 13, 853–861 47. Mitchell, T.J. and Dalziel, C.E. (2014) The biology of pneumolysin. Subcell. Biochem. 80, 145–160
23. Ring, A. et al. (1998) Pneumococcal trafficking across the blood– brain barrier. Molecular analysis of a novel bidirectional pathway. J. Clin. Invest. 102, 347–360
48. Marriott, H.M. et al. (2008) Pneumolysin: a double-edged sword during the host-pathogen interaction. Curr. Mol. Med. 8, 497–509
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26. Honda, Z. et al. (2002) Platelet-activating factor receptor. J. Biochem. 131, 773–779 27. Cundell, D.R. et al. (1995) Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor. Nature 377, 435–438 28. Radin, J.N. et al. (2005) beta-Arrestin 1 participates in plateletactivating factor receptor-mediated endocytosis of Streptococcus pneumoniae. Infect. Immun. 73, 827–835 29. Iovino, F. et al. (2014) Streptococcus pneumoniae interacts with pIgR expressed by the brain microvascular endothelium but does not co-localize with PAF receptor. PLoS ONE 9, e97914 30. Wang, K. et al. (1992) High affinity laminin receptor is a receptor for Sindbis virus in mammalian cells. J. Virol. 66, 4992–5001
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51. Hikiji, H. et al. (2004) Absence of platelet-factor receptor protects mice from osteoporosis following ovariectomy. J. Clin. Invest. 114, 85–93 52. Marina, Z. (2004) Absence of PAF actions increases angiogenesis. Br. J. Pharmacol. 141, 1085–1086 53. Finn-Eirik and Johansen et al. (1999) Absence of epithelial immunoglobulin a transport, with increased mucosal leakiness, in polymeric immunoglobulin receptor/secretory component-deficient mice. J. Exp. Med. 190, 915–922 54. Duncan, G.S. et al. (1999) Genetic evidence for functional redundancy of Platelet/Endothelial cell adhesion molecule-1 (PECAM-1): CD31-deficient mice reveal PECAM-1-dependent and PECAM-1independent functions. J. Immunol. 162, 3022–3030
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Review
How Does Streptococcus pneumoniae Invade the Brain? Federico Iovino,1,2,* Jolien Seinen,3 Birgitta Henriques-Normark,1,2,4 and Jan Maarten van[15_TD$IF] Dijl3,4 Streptococcus pneumoniae (the pneumococcus) is the major cause of bacterial meningitis. The mechanisms by which pneumococci from the bloodstream penetrate the blood–brain barrier to reach the brain are not fully understood. Receptor-mediated adhesion of the bacteria to the brain endothelium is considered a key event leading to meningitis development. The aim of this review is to discuss recent advances and perspectives related to the interactions of S. pneumoniae with the blood–brain barrier during the events leading to meningitis. Altogether, the available data suggest that, by precisely defining the pathways and ligands by which S. pneumoniae adheres to specific receptors, it may be possible to interfere with the respective mechanisms and develop strategies to prevent or even cure pneumococcal meningitis. Streptococcus pneumoniae, the Blood–Brain Barrier, and Pneumococcal Meningitis The Gram-positive bacterium [26_TD$IF]Streptococcus pneumoniae (the pneumococcus) is an important commensal resident of the human nasopharynx. Although carriage is usually asymptomatic, S. pneumoniae can become invasive and spread from the upper respiratory tract to other organs, leading to serious diseases such as pneumonia, sepsis, or meningitis [1,2]. S. pneumoniae is the major etiological cause of bacterial meningitis, responsible for two-thirds of meningitis cases in Europe and in the USA [3–5]. Bacterial meningitis is a disease with high morbidity and mortality worldwide despite the implementation of several vaccination programs and antimicrobial agents [3–6]. A major route for bacteria to reach the meninges is through the bloodstream [7]. Having reached the blood vessels in the brain, bacteria present in the bloodstream have to cross the blood–brain barrier to enter the brain and cause infection. This view is supported by recent immunofluorescent analyses combined with high-resolution confocal microscopy, where blood-borne S. pneumoniae was clearly shown to adhere to the brain vascular endothelium prior to invasion of the brain [8]. The blood–brain barrier is a specialized vasculature system that separates the brain from circulating blood and has critical functions in both protection and nutrient supply of the brain [9–11]. Endothelial cells form the layer that lines the interior surface of the blood vessels [12,13]. Pathogens can invade the brain only after crossing the endothelial cell layer of the blood–brain barrier and, therefore, they must develop strategies to pass this barrier. The inflammatory features and clinical complications following bacterial meningitis normally observed in humans can be reproduced using animal models. Notably, most in vivo models that examine the pathophysiology of bacterial meningitis involve the direct injection of pneumococci into the brain of mice or rats [14–18]. Administration of bacteria directly into the brain bypasses the need for blood–brain barrier translocation. In order to study the interaction of blood-borne
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Trends Streptococcus pneumoniae, the main causative agent of bacterial meningitis, penetrates the blood–brain barrier by binding to PECAM-1 and pIgR expressed by brain vascular endothelial cells. Overall, recent findings suggest that PECAM-1 and pIgR, together with platelet-activating factor receptor (PAFR), another receptor thought to be more involved in signaling rather than physical binding, might cooperate during adhesion of pneumococci to brain endothelial cells. The passage of S. pneumoniae across the blood–brain barrier is a crucial step for meningitis development. The knowledge of how S. pneumoniae penetrates the blood–brain barrier can be used to develop strategies aimed at interfering with the respective pathways in order to prevent the entry of the pathogen into the brain.
1 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE 171 77, Stockholm, Sweden 2 Department of Clinical Microbiology, Karolinska University Hospital, SE 171 76, Stockholm, Sweden 3 Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 4 These authors contributed equally to this work.
*Correspondence: [email protected] (F. Iovino).
http://dx.doi.org/10.1016/j.tim.2015.12.012 © 2016 Elsevier Ltd. All rights reserved.
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S. pneumoniae with the brain vascular endothelium in vivo, prior to the onset of meningitis, a socalled bacteremia-derived meningitis model was established in recent years [8,19]. This model, where bacteria are injected into the bloodstream instead of the brain, has allowed the visualization of successive steps from bacteremia to brain invasion. The aim of the present review is to discuss recent advances in our understanding how blood-borne S. pneumoniae interacts with the blood–brain barrier endothelium during the events leading up to meningitis, and how to use such knowledge to develop new therapeutic strategies to fight the disease.
Receptor-Mediated Transcytosis How bacterial pathogens cross the blood–brain barrier is still subject [27_TD$IF]of debate. Several possible mechanisms have been implicated in this process, such as: (i) destruction of the endothelial cell layers in case of, for example, Neisseria meningitidis [20]; (ii) traversal of the blood–brain barrier in between the endothelial cells by disruption of tight junctions in case of group A streptococci [21]; and (iii) receptor-mediated transcytosis across endothelial and epithelial cell layers in case of S. pneumoniae [22,23]. The view that pneumococci pass the blood–brain barrier via transcytosis rather than pericellularly is supported by experiments, showing a continuous and homogeneous staining of VE-Cadherin in the brain of infected mice [8]. This implies that blood-borne S. pneumoniae does not cause a disruption of the endothelial tight junctions. In this context, it should be noted that bacterial pathogens such as pneumococci have the capability to bind to certain receptors on the plasma membrane of epithelial and endothelial cells, and this receptormediated binding facilitates bacterial invasion into[3_TD$IF] and translocation over[3_TD$IF] human cell layers. The passage of bacterial pathogens across such layers is a fundamental step for development of invasive diseases and it is necessary for S. pneumoniae to cross the blood–brain barrier to develop bacterial meningitis [22,23]. The Platelet-Activating Factor Receptor (PAFR) The first reported receptor implicated in adhesion to, invasion of, and also transcytosis through[3_TD$IF] endothelial cells is the [28_TD$IF]PAFR[6_TD$IF]. PAFR is a G-protein-coupled receptor with seven transmembrane domains and its natural ligand is the platelet-activating factor (PAF). PAF is a mediator in diverse pathologic processes, such as allergy, asthma, septic shock, arterial thrombosis, and inflammation [24–26]. Binding of PAF to the PAFR stimulates numerous signal transduction pathways including phospholipase C, D, A2, mitogen-activated protein kinases (MAPKs), and the phosphatidylinositol-calcium second messenger system [25]. PAFR has been proposed to bind S. pneumoniae, thereby facilitating adhesion to, uptake by, and transcytosis through[3_TD$IF] endothelial cells leading to invasive disease [23,27,28]. However, while in vitro and in vivo studies indicate that PAFR is involved in the development of invasive pneumococcal disease (IPD), there is so far no unequivocal published evidence that a direct binding between S. pneumoniae and PAFR occurs [24]. A recent study suggests that PAFR plays a role in pneumococcal adhesion to endothelial cells, even though pneumococci do not directly bind to PAFR [29]. After immunofluorescent microscopy analysis, colocalization of PAFR and S. pneumoniae was not observed in vivo in mouse brain tissue nor in endothelial cells in vitro. Nevertheless, upon blockade of PAFR, adhesion of pneumococci to human endothelial cells was significantly reduced, indicating that PAFR most likely has an indirect role in IPD [29]. When the PAFR was genetically deleted or chemically inhibited, S. pneumoniae was still able to adhere to human cells in vitro, and to cause invasive disease in vivo [23,27]. The Laminin Receptor (LR) The [29_TD$IF]LR[6_TD$IF] is an important molecule involved both in cell adhesion to the basement membrane and in signal transduction following this binding event. The LR on endothelial cells was found to interact with neurotropic viruses, including Sindbis virus [30], Dengue virus [31], adenoassociated virus [32], tick-borne encephalitis virus, and Venezuelan equine encephalitis virus [33]. LR was identified to be a common receptor for both S. pneumoniae and N. meningitidis on
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the surface of rodent and human brain microvascular endothelial cells [19]. Fluorescent beads coated with the pneumococcal choline-binding protein (Cbp) A [34–36] that were injected intravascularly into mice adhered to the cerebral endothelium. Pretreatment with anti-LR antibody led to an inhibition of the adherence of the CbpA–beads to the endothelium in mice, thus suggesting that the LR interacts with the bacteria[3_TD$IF] facilitating translocation of intracellular pneumococci across the blood–brain barrier endothelium, from the basolateral side of endothelial cells to the brain tissue [19]. The Poly Immunoglobulin Receptor (pIgR) The [30_TD$IF]pIgR[6_TD$IF] transports immunoglobulins across mucosal epithelial barriers (e.g., in the respiratory tract) as a first line of defense to antigens from pathogenic bacteria [37–39]. S. pneumoniae has been shown to bind to pIgR expressed by human nasopharyngeal epithelial cells[3_TD$IF] and this binding facilitates pneumococcal translocation through the nasopharyngeal epithelium [22]. When human nasopharyngeal epithelial (Detroit) cells were treated with an antibody against pIgR prior to pneumococcal challenge, adherence was reduced [22]. This implies that pIgR is functionally involved in pneumococcal adhesion to nasopharyngeal epithelium. Furthermore, immunoblotting analyses showed that pneumococcal CbpA binds to human pIgR, indicating that CbpA may be required to mediate binding of S. pneumoniae to this receptor [22]. PIgR is not only present on the epithelium of the respiratory tract, but can also be found on the plasma membrane of endothelial cells, especially brain endothelial cells [29]. Due to its presence on the blood–brain barrier endothelium, the possible involvement of pIgR in pneumococcal adhesion to brain endothelial cells was recently investigated [29]. Notably, immunofluorescent analyses of brain tissue of infected mice showed that most pneumococci adhering to the brain vasculature were colocalized with pIgR [29]. Furthermore, by incubating pneumococci with human endothelial cell lysates, it was shown that S. pneumoniae can directly bind to pIgR [29]. The combined results therefore suggest that pIgR is a direct adhesion receptor for S. pneumoniae on the blood–brain barrier endothelium. Even though the main route of brain invasion seems to be the bloodstream, pneumococci may also reach the brain through skull fractures that tear the dura mater, or directly from the nasopharynx. In fact, it was recently reported that S. pneumoniae can transmigrate from the nasopharyngeal epithelium to the olfactory nerves that anatomically connect the upper respiratory tract with the central nervous system [40]. Although it was not shown experimentally, an intriguing hypothesis is that the pIgR on the nasopharyngeal epithelium could mediate the passage of the bacteria from the epithelial cells to the olfactory nerve. Clearly, once this passage has occurred the bacteria can travel along the nerve and reach the brain. Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) The [31_TD$IF]PECAM-1 ([10_TD$IF]also known as CD31) is a pan-endothelial protein[3_TD$IF] present in the intercellular junctions of the endothelial cells, that is involved in leukocyte migration, angiogenesis, and integrin activation [41–43]. In particular, the involvement of PECAM-1 in leukocyte–endothelium interaction and leukocyte trans-endothelial migration in the vascular system makes PECAM-1 a key molecule in inflammation and neuroinflammation [43]. Recently, PECAM-1 was implicated in gastrointestinal infection caused by Salmonella enterica serovar Typhimurium [44], suggesting an active role of PECAM-1 in host–pathogen interactions. This raised the question whether PECAM-1 might also play a role in host–pneumococcal interactions. Indeed, for the first time it was recently shown that, in brain tissue from intravenously infected mice, most pneumococci did colocalize with PECAM-1 [45]. Blockade of PECAM-1 with a specific antibody in human brain endothelial cells led to a significant reduction of bacterial adhesion, indicating that PECAM-1 is functionally involved in S. pneumoniae adhesion to the brain vascular endothelium [45]. Using a biochemical approach, it was demonstrated that S. pneumoniae can directly bind PECAM-1 expressed by human endothelial cells [45]. Importantly, PECAM-1 colocalized with pIgR on the
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Key Figure
Cooperation of Platelet-Activating Factor Receptor (PAFR), pIgR, and PECAM-1 in Pneumococcal Adhesion to Endothelial Cells: A Proposed New Model (A)
(B) S. pneumoniae
Inflammation
Inflammatory cytokines
Pneumococcal PAF components
plgR
S. pneumoniae binding sites
PAFR before acvaon
More S. pneumoniae binding sites
More adhering pneumococci
PAFR activation PAFR
plgR
Blood
plgR-PECAM-1 upregulation
Endothelial cell
Brain
Figure 1. (A) Under normal physiological conditions, PAFR is present on the plasma membrane of endothelial cells in its non-activated state, while pIgR and PECAM-1 are forming a double receptor displaying binding sites that can potentially be used as anchors for the adhesion of bacterial pathogens.[1_TD$IF] (B) Upon pneumococcal infection, inflammation develops and PAFR is activated by pneumococcal components, such as pneumolysin and choline-binding proteins, and PAF. Activation of PAFR may lead to the upregulation of pIgR–PECAM-1. This enhanced expression of pIgR–PECAM-1 would make more potential binding sites available for Streptococcus pneumoniae and, ultimately, this would lead to more pneumococci adhering to endothelial cells, allowing them to enter the brain.
brain endothelium and, moreover, the two receptors directly interacted with each other as demonstrated by co-immunoprecipitation experiments suggesting the possible formation of a double receptor that facilitates the passage of S. pneumoniae across the blood–brain barrier [45] (Figure 1, Key Figure). Nevertheless, to unequivocally demonstrate the joint role of PECAM-1 and pIgR in pneumococcal meningitis, S. pneumoniae should be intravenously administered to PECAM-1 / [2_TD$IF] and pIgR / mice. In the case of a direct role, as suspected, one would expect that only few bacteria can translocate the blood–brain barrier in the knock-out mice. Although current literature still lacks such experimental evidence, it was previously shown that adhesion of bacteria to the brain vasculature endothelium represents a crucial moment for invasion of meningeal pathogens in the brain. In addition, the observation that blood-borne pneumococci colocalize with both PECAM-1 and pIgR in vivo strongly indicates that the two receptors jointly facilitate the passage of the bacteria from the bloodstream into the brain [29,45].
Cooperation of PAFR, pIgR and PECAM-1 during Pneumococcal Adhesion to Endothelial Cells: A New Model From the current literature [24], it remains unclear what the exact role of PAFR is in IPD. The involvement of PAFR in S. pneumoniae adhesion to endothelial cells might well be exerted through the binding of PAF or pneumococcal cell-wall components to the receptor and the subsequent inflammatory consequences of its activation [24]. Inflammation leads to an upregulation of receptors expressed on the plasma membrane of the apical side of the cells [7,27,46], and PECAM-1 and pIgR may be among them. In the presence of more pIgR and PECAM-1,
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S. pneumoniae has more sites to adhere to on the endothelial cells. It has been repeatedly shown that blocking PAFR with a specific antibody or antagonist leads to a significant reduction of pneumococcal adhesion to endothelial cells in vitro [23,27,29]. We therefore hypothesize that, due to PAFR blocking, the upregulation of pIgR and PECAM-1 may be prohibited. Possibly, the presence of pneumococci may trigger the autocrine production of PAF, directly or indirectly, which activates PAFR, leading to an upregulation of pIgR and PECAM-1. This would increase pneumococcal adherence to pIgR and PECAM-1 and, in turn, more adherent bacteria would increase the chances of bacterial invasion into the cells (Figure 1). Whether PAFR activation leads, directly or indirectly, to pIgR-PECAM-1 upregulation still remains an open question. Besides PAF, secreted pneumococcal components, such as pneumolysin [47,48] and CbpA, could bind to PAFR and activate this receptor [49]. Clearly, this idea still needs to be experimentally validated. Accordingly, an in-depth investigation of secreted and surface-attached pneumococcal proteins, including proteomics and structural analyses, will be required to identify any protein(s) of S. pneumoniae that could be recognized and bound by PAFR. Interesting candidate proteins for such studies were recently identified by Pribyl et al. in a proteomics-based survey of the pneumococcal surface-bound and secreted proteins [50]. An important recent finding was that if pIgR and PECAM-1 are blocked simultaneously, pneumococcal adhesion to endothelial cells is reduced even more than when the two receptors are blocked individually [45]. Bacteria cannot adhere to pIgR and PECAM-1 after blocking with their respective antibodies, but PAFR activation may still be possible and could thus lead to upregulation of PECAM-1 and pIgR expression.
Humans Deficient in Receptors PAFR, pIgR, and PECAM-1 Have Not Been Identified, but Mice Survive without Them Humans deficient in PAFR have not been identified. However PAFR–/– mice have been generated [51]. In PAFR–/– mice, modifications of known angiogenic factors, such as vascular endothelial growth factor (VEGF), have not been observed, but a sustained rise in the chemokines CXCL2 and CCL2 has been detected, which may compensate for the absence of PAFR as an important molecule in maintaining immunological surveillance [52]. These findings indicate that the absence of PAFR in mice is compatible with health, although it has to be noted that this was observed under controlled conditions. Mice lacking pIgR were of normal size and fertility as well, but displayed increased IgG levels in their sera, suggesting a triggering of systemic immunity [53]. These findings indicate that absence of pIgR is not lethal either, but at the same time it seems that absence of pIgR is sensed as a potential danger by the systemic immune system [38,53]. PECAM-1 is expressed not only by endothelial cells but also by platelets, neutrophils, and macrophages, and its signaling pathways might mediate resistance to apoptosis and promote cell survival [41,42]. Absence of PECAM-1 may therefore lead to several cellular dysfunctions. Interestingly, PECAM-1 was reported not to be required for embryogenesis, fetal maturation, or fertility in mice, and, furthermore, PECAM-1 expression on platelets is not essential for platelet–platelet aggregation [54]. PECAM-1–/– animals develop normally and do not show any signs of an abnormal physiology under normal baseline conditions. However, as the animals age they can develop an autoimmune lupus-like syndrome and, when stressed, a variety of abnormal responses have also been noticed. In particular, PECAM-1–/– animals exhibited a prolonged bleeding time [55]. Thus, although the absence of the PAFR, pIgR, or PECAM-1 receptors did not cause serious problems in mice during certain circumstances, dysfunctions and abnormalities due to a lack of these receptors cannot be excluded.
Blockade of S. pneumoniae Receptors: A New Avenue for the Prevention and Cure of Pneumococcal Meningitis Knowledge of the mechanisms by which the pneumococcus interacts with the blood–brain barrier before invading the brain is fundamental in order to develop therapeutic strategies to avoid adhesion of S. pneumoniae to the blood–brain barrier and, thus, invasion of the pathogen
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into the central nervous system. Adhesion of pneumococci to the blood–brain barrier is spatiotemporally controlled at different sites throughout the brain, and the local immune system in the brain is activated immediately as soon as bacteria in the bloodstream interact with the blood–brain barrier [8]. The bacteremia-derived meningitis model is used to investigate the interactions of bacteria with the blood–brain barrier, including the events preceding meningitis [8,19,29,45]. To study meningitis itself, intracisternal administration of bacteria was previously used to induce meningitis in vivo [14–18]. Using this model, neutrophil influx into the brain was detected only at 30 hours after challenge, indicating that white blood cells can be detected in the brain only at late stages of the disease [14]. A high count of white blood cells in the cerebrospinal fluid (CSF) is often used as a clinical test for diagnosis of meningitis [56]. Moreover, classic symptoms of meningitis, such as rash, neck stiffness, photophobia, severe headache, and impaired consciousness[3_TD$IF] develop late [56–58]. While these symptoms represent the classical picture of suspected meningitis, the disease may be already at an advanced stage in patients displaying them. Thus, it is not really surprising that, at this stage, the risks of brain damage are considerable even if the infection is successfully fought with antibiotics. World Health Organization (WHO) data indicate that, even if the disease is diagnosed early and adequate treatment is started immediately, 5–10% of the patients will die, typically within 24[32_TD$IF]–48 hours after occurrence of the first symptoms. When left untreated, up to 50% of the patients may die. In case of recovery, brain damage, hearing loss, or a learning disability appears in 10–20% of the survivors (http://www.who.int/topics/meningitis/en/). It is therefore crucial to start interventions before bacteria have invaded the brain in order to optimize the survival chances of patients and to prevent permanent damage. Treatment with antibiotics is the clinically used way to cure meningitis, and routine vaccination against pneumococcal infections with the pneumococcal conjugate vaccine (PCV), which is active against seven to thirteen common capsular serotypes of this pathogen, has significantly reduced the incidence of invasive pneumococcal disease in vaccinated children [59–61]. Knowing that PAFR, pIgR, and PECAM-1 mediate the first adhesion of blood-borne S. pneumoniae to the vascular endothelium of the blood–brain barrier opens a complementary approach to antibiotics and vaccines for curing or preventing pneumococcal meningitis by developing therapies that interfere with these receptors. This could, for example, be achieved by blocking PAFR, pIgR, and PECAM-1 with specific antibodies which would limit the adhesion of pneumococci to the blood–brain barrier and then observing whether the systemic administration of blocking antibodies leads to less severe disease symptoms. Alternatively, blockade of the receptors can be achieved by using either chemical antagonists, such as, for instance, L659989 or WEB2086 previously used to successfully block the PAFR in vitro [24], or the pneumococcal proteins that should specifically anchor to the receptors, such as CbpA that was reported to bind to pIgR in the respiratory epithelium [22,62]. Another alternative, but technically more challenging approach, is to prevent binding of S. pneumoniae to PAFR, pIgR, and PECAM-1 on the blood–brain barrier endothelium by blocking the respective ligands on the pneumococcal cell surface with engineered soluble derivatives of these receptors. This would have the potential advantage of minimal interference with the physiological functions of these three receptors. Such studies could be aided by targeted infection-imaging approaches, for instance, with positron emission tomography (PET) tracers that bind well to S. pneumoniae [63]. However, while blocking of these pneumococcal receptors could provide protection against meningitis, it will be crucial to develop strategies to avoid possible dysfunctions caused by their blocking before this approach can be implemented in the clinic. Based on literature data, mutated PAFR in mice is not likely to cause serious dysfunctions [51]. However, mutated pIgR may interfere with the normal transport of immunoglobulins across cells. It has been shown that treatment with phorbol myristyl acetate (PMA) activates isozymes of protein kinase C (PKC), a downstream protein in the signaling cascade of pIgR [[3_TD$IF]64]. This activation stimulates not only pIgR transcytosis, but more generally the pathway of a variety of molecules in the transcytotic pathways [[3_TD$IF]64]. Thus, treatment with PMA can stimulate cells to use other transcytotic systems and their receptors, in case immunoglobulin transfer mediated by pIgR is altered. In this way
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transport of immunoglobulins can be maintained through other transcytotic pathways. Regarding the vascular role of PECAM-1, it is important to bear in mind that endothelial cells express a vast range of endothelial adhesion factors (such as VECAM-1 and ICAM-1) that perform similar functions as PECAM-1 [[34_TD$IF]65,66]. Therefore, is conceivable that such adhesion molecules could complement for PECAM-1's role in case PECAM-1 is blocked. In principle, it should thus be possible to develop monoclonal antibodies blocking the pneumococcal binding site of the PECAM-1 receptor. While the blocking of pIgR and PECAM-1 showed only a partial reduction of pneumococcal adherence to endothelial cells [45], the blocking of the PAFR, pIgR, and PECAM-1 receptors at the same time could be a potential way to completely preclude the adhesion of S. pneumoniae to the blood–brain barrier endothelium and, therefore, prevent completely pneumococcal invasion into the brain. This, however, remains to be assessed experimentally. Clearly, an important advantage of conceivable therapies based on monoclonal antibodies against PAFR, pIgR, and PECAM-1 would be that these antibodies need to be administered only as long as pneumococci are detectable in the blood. Once these have been fought successfully with regular antibiotics, the antibody therapy can be stopped, which will limit possible unwanted side effects to a minimum.
Outstanding Questions How exactly does the signaling receptor PAFR cooperate with the binding receptors pIgR and PECAM-1 during the adhesion of S. pneumoniae to brain endothelial cells? Does an absence of adhesion receptors lead to less bacterial invasion into the brain in animal meningitis models? Is the treatment with blocking antibodies/antagonists/receptor ligands a valid way to block the adhesion receptors and prevent bacterial passage across the blood–brain barrier? If so, can such treatment be applied in humans?
Lastly, it has been demonstrated that pneumococci in the blood can reach and adhere to the blood–brain barrier endothelium after which they may invade the brain [8]. Bacteremia is present in 20–30% of cases with pneumococcal pneumonia, which can occur when an infection of the lungs grows out of control [[35_TD$IF]67–[36_TD$IF]69]. Patients with bacteremia and pneumonia can be considered ‘high-risk’ for progression towards brain infection and, for this reason, it could be valuable for them to receive innovative treatments that block pneumococcal receptors. This would prevent pneumococci from adhering to the blood–brain barrier and invading the brain. Such a treatment would even be of relevance for patients who are hospitalized with meningitis as it would still reduce the numbers of bacteria adhering to the blood–brain barrier and, in combination with antibiotic therapy, both the blood-borne bacteria and the bacteria that have already reached the brain could be eliminated.
Concluding Remarks What is the ultimate goal of knowing how S. pneumoniae invades the brain? Clearly, if pneumococci cannot adhere to endothelial cells, bacterial invasion will most likely be prevented. Therefore, by precisely defining the pathways by which S. pneumoniae adheres to specific receptors, it may be possible to develop drugs that interfere with these mechanisms and to develop strategies to prevent or even cure pneumococcal meningitis, as this still remains a very serious disease with high mortality and morbidity worldwide[37_TD$IF] (see Outstanding Questions). Judged by our current understanding of pneumococcal brain invasion, this is a highly ambitious but potentially achievable objective. Acknowledgments We thank Staffan Normark, Jetta Bijlsma and Ingrid Molema for their critical feedback[38_TD$IF]. F.I. and B.H.-N. were supported by grants from the Swedish Research Council, ALF grant from Stockholm County Council, the Foundation for Strategic Research (SSF), and Knut and Alice Wallenberg foundation. F.I. and J.S. were supported in part by grants from the Graduate School of Medical Sciences of the University of Groningen. J.S. and J.M.v.D. acknowledge the Deutsche Forschungsgemeinschaft Research Training Group GRK1870 for support.
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