Pericytes are protective in experimental pneumococcal meningitis through regulating leukocyte infiltration and blood-brain barrier function.

BACKGROUND: Brain pericytes participate in the regulation of cerebral blood flow and the maintenance of blood-brain barrier integrity. Because of their perivascular localization, their receptor repertoire, and their potential ability to respond to inflammatory and infectious stimuli by producing various cytokines and chemokines, these cells are also thought to play an active role in the immune response to brain infections. This assumption is mainly supported by in vitro studies, investigations in in vivo disease models are largely missing. Here, we analysed the role of brain pericytes in pneumococcal meningitis, in vitro and in vivo in two animal models of pneumococcal meningitis. METHODS: Primary murine and human pericytes were stimulated with increasing concentrations of different serotypes of Streptococcus pneumoniae in the presence or absence of Toll-like receptor inhibitors and their cell viability and cytokine production were monitored. To gain insight into the role of pericytes in brain infection in vivo, we performed studies in a zebrafish embryo model of pneumococcal meningitis in which pericytes were pharmacologically depleted. Furthermore, we analyzed the impact of genetically induced pericyte ablation on disease progression, intracranial complications, and brain inflammation in an adult mouse model of this disease. RESULTS: Both murine and human pericytes reacted to pneumococcal exposure with the release of selected cytokines. This cytokine release is pneumolysin-dependent, TLR-dependent in murine (but not human) pericytes and can be significantly increased by macrophage-derived IL-1b. Pharmacological depletion of pericytes in zebrafish embryos resulted in increased cerebral edema and mortality due to pneumococcal meningitis. Correspondingly, in an adult mouse meningitis model, a more pronounced blood-brain barrier disruption and leukocyte infiltration, resulting in an unfavorable disease course, was observed following genetic pericyte ablation. The degree of leukocyte infiltration positively correlated with an upregulation of chemokine expression in the brains of pericyte-depleted mice. CONCLUSIONS: Our findings show that pericytes play a protective role in pneumococcal meningitis by impeding leukocyte migration and preventing blood-brain barrier breaching. Thus, preserving the integrity of the pericyte population has the potential as a new therapeutic strategy in pneumococcal meningitis.

The role of plasminogen activator inhibitor-2 in pneumococcal meningitis.

Pneumococcal meningitis is associated with dysregulation of the coagulation cascade. Previously, we detected upregulation of cerebral plasminogen activator inhibitor-2 (PAI-2) mRNA expression during pneumococcal meningitis. Diverse functions have been ascribed to PAI-2, but its role remains unclear. We analyzed the function of SERPINB2 (coding for PAI-2) in patients with bacterial meningitis, in a well-established pneumococcal meningitis mouse model, using Serpinb2 knockout mice, and in vitro in wt and PAI-2-deficient bone marrow-derived macrophages (BMDMs). We measured PAI-2 in cerebrospinal fluid of patients, and performed functional, histopathological, protein and mRNA expression analyses in vivo and in vitro. We found a substantial increase of PAI-2 concentration in CSF of patients with pneumococcal meningitis, and up-regulation and increased release of PAI-2 in mice. PAI-2 deficiency was associated with increased mortality in murine pneumococcal meningitis and cerebral hemorrhages. Serpinb2-/- mice exhibited increased C5a levels, but decreased IL-10 levels in the brain during pneumococcal infection. Our in vitro experiments confirmed increased expression and release of PAI-2 by wt BMDM and decreased IL-10 liberation by PAI-2-deficient BMDM upon pneumococcal challenge. Our data show that PAI-2 is elevated during in pneumococcal meningitis in humans and mice. PAI-2 deficiency causes an inflammatory imbalance, resulting in increased brain pathology and mortality.

Microglial response in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice after systemic stimulation with Escherichia coli.

BACKGROUND: Systemic infection is an important risk factor for delirium, associated with neurodegeneration and subsequent cognitive impairment in older people. Microglial cell response is a known key player in this process and we hypothesize that the triggering receptor expressed on myeloid cells 2 (TREM2) plays an important role in the regulation of this response. METHODS: 8- to 10-week old male wild-type (WT) and TREM2 knock-out (Trem2-/-) mice were intraperitoneally inoculated with live Escherichia coli (E. coli) or saline. After inoculation, all mice were treated with ceftriaxone (an antimicrobial drug) at 12 and 24 h and were sacrificed after 2 and 3 days. Microglial response was determined by immunohistochemical staining with an ionized calcium-binding adaptor molecule 1 (Iba-1) antibody and flow cytometry. mRNA expression of pro- and anti-inflammatory mediators was measured to quantify the inflammatory response. RESULTS: We observed increased Iba-1 positive cells number in thalamus of Trem2-/- mice at 3d after inoculation compared to WT mice (mean 120 cell/mm2 [SD 8] vs 105 cell/mm2 [SD 11]; p = 0.03). Flow cytometry showed no differences in forward scatter or expression of CD11b, CD45 and CD14 between WT and Trem2-/- mice. The brain mRNA expression levels of tumor necrosis factor alpha (TNF-α) of Trem2-/- mice at 2d were higher compared to WT mice (p = 0.003). Higher mRNA expression of interleukin 1 beta (IL-1ß), Iba-1, CD11b and mitogen-activated protein kinase 1 (MAPK-1) was found in brain of WT mice at 2d compared to Trem2-/- mice (respectively p = 0.02; p = 0.001; p = 0.03 and p = 0.02). In spleen there were no differences in inflammatory mediators, between WT and Trem2-/- mice. INTERPRETATION: Although the loss of function of TREM2 during systemic infection led to an increased number of activated microglia in the thalamus, we did not observe a consistent increase in expression of inflammatory genes in the brain. The role of TREM2 in the neuro-inflammatory response following systemic infection therefore appears to be limited.

Microglial cell response in α7 nicotinic acetylcholine receptor-deficient mice after systemic infection with Escherichia coli.

BACKGROUND: Development of neurodegeneration in older people has been associated with microglial cell activation triggered by systemic infection. We hypothesize that α7 nicotinic acetylcholine receptor (α7nAChR) plays an important role in regulation of this process. METHODS: 8- to 10-week-old male wild-type (WT) and α7nAChR knock-out (α7nAChR-/-) mice were intraperitoneally inoculated with live Escherichia (E.) coli or saline. After inoculation, all mice were treated with ceftriaxone (an antimicrobial drug) at 12 and 24 h and killed at 2 or 3 days. The microglial response was characterized by immunohistochemical staining with an ionized calcium-binding adaptor molecule 1 (Iba-1) antibody and flow cytometry. To quantify inflammatory response, mRNA expression of pro- and anti-inflammatory mediators was measured in brain and spleen. RESULTS: We observed no differences in Iba-1 positive cell number or morphology and flow cytometry (CD11b, CD45 and CD14) of microglial cells between WT and α7nAChR-/- mice after systemic infection. Infected α7nAChR-/- mice showed significantly higher mRNA expression in brain for tumor necrosis factor alpha (TNF-α) at day 2 and 3, interleukin 6 (IL-6) at day 2 and monocyte chemotactic protein 1 (MCP-1) and suppressor of cytokine signaling 1 (SOCS1) at day 3, there was significantly lower mRNA expression in brain for mitogen-activated protein kinase 1 (MAPK1) at day 2 and 3, high-mobility group 1 (HMGB-1) and CD11b at day 2, and deubiquitinase protein A20 (A20) at day 3 compared to infected WT mice. INTERPRETATION: Loss of function of α7nAChR during systemic infection led to an increased expression of TNF-α and IL-6 in brain after systemic infection with E. coli, but not to distinct differences in microglial cell number or morphological activation of microglia.

Metabolic rerouting via SCD1 induction impacts X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease caused by mutations in ABCD1, the peroxisomal very long-chain fatty acid (VLCFA) transporter. ABCD1 deficiency results in accumulation of saturated VLCFAs. A drug screen using a phenotypic motor assay in a zebrafish ALD model identified chloroquine as the top hit. Chloroquine increased expression of stearoyl-CoA desaturase-1 (scd1), the enzyme mediating fatty acid saturation status, suggesting that a shift toward monounsaturated fatty acids relieved toxicity. In human ALD fibroblasts, chloroquine also increased SCD1 levels and reduced saturated VLCFAs. Conversely, pharmacological inhibition of SCD1 expression led to an increase in saturated VLCFAs, and CRISPR knockout of scd1 in zebrafish mimicked the motor phenotype of ALD zebrafish. Importantly, saturated VLCFAs caused ER stress in ALD fibroblasts, whereas monounsaturated VLCFA did not. In parallel, we used liver X receptor (LXR) agonists to increase SCD1 expression, causing a shift from saturated toward monounsaturated VLCFA and normalizing phospholipid profiles. Finally, Abcd1-/y mice receiving LXR agonist in their diet had VLCFA reductions in ALD-relevant tissues. These results suggest that metabolic rerouting of saturated to monounsaturated VLCFAs may alleviate lipid toxicity, a strategy that may be beneficial in ALD and other peroxisomal diseases in which VLCFAs play a key role.

Aging and Microglial Response following Systemic Stimulation with Escherichia coli in Mice.

Systemic infection is an important risk factor for the development cognitive impairment and neurodegeneration in older people. Animal experiments show that systemic challenges with live bacteria cause a neuro-inflammatory response, but the effect of age on this response in these models is unknown. Young (2 months) and middle-aged mice (13-14 months) were intraperitoneally challenged with live Escherichia coli (E. coli) or saline. The mice were sacrificed at 2, 3 and 7 days after inoculation; for all time points, the mice were treated with ceftriaxone (an antimicrobial drug) at 12 and 24 h after inoculation. Microglial response was monitored by immunohistochemical staining with an ionized calcium-binding adaptor molecule 1 (Iba-1) antibody and flow cytometry, and inflammatory response by mRNA expression of pro- and anti-inflammatory mediators. We observed an increased microglial cell number and moderate morphologically activated microglial cells in middle-aged mice, as compared to young mice, after intraperitoneal challenge with live E. coli. Flow cytometry of microglial cells showed higher CD45 and CD11b expressions in middle-aged infected mice compared to young infected mice. The brain expression levels of pro-inflammatory genes were higher in middle-aged than in young infected mice, while middle-aged infected mice had similar expression levels of these genes in the systemic compartment. We conclude that systemic challenge with live bacteria causes an age-dependent neuro-inflammatory and microglial response. Our data show signs of an age-dependent disconnection of the inflammatory transcriptional signature between the brain and the systemic compartment.

Cerebral venous thrombosis in pneumococcal meningitis: An autopsy study.

AIMS: Cerebral venous thrombosis (CVT) is a rare but severe complication of bacterial meningitis. The histopathological features of CVT in meningitis patients have not been described. MATERIALS AND METHODS: We studied histopathology findings of brain autopsy material from 2 patients with bacterial meningitis complicated by CVT and compared findings with those in 3 CVT patients without meningitis and 1 patient with bacterial meningitis without CVT. The histological slides were re-evaluated and assessed for the presence of thrombosis, cerebral venous sinus mural inflammation and bleeding, inflammation at the thrombosis attachment point, endothelial abnormalities, and the presence of bacteria. RESULTS: The 2 patients who died of bacterial meningitis complicated by CVT showed multifocal deep intramural inflammation in the cerebral venous sinus, whereas this was absent in patients with only bacterial meningitis or CVT. Bacteria were identified within the intramural inflammation and thrombus. CONCLUSION: We observed bacterial invasion causing multifocal deep intramural inflammation and venous wall disintegration as CVT in pneumococcal meningitis.

Complement factor H contributes to mortality in humans and mice with bacterial meningitis.

BACKGROUND: The complement system is a vital component of the inflammatory response occurring during bacterial meningitis. Blocking the complement system was shown to improve the outcome of experimental pneumococcal meningitis. Complement factor H (FH) is a complement regulatory protein inhibiting alternative pathway activation but is also exploited by the pneumococcus to prevent complement activation on its surface conferring serum resistance. METHODS: In a nationwide prospective cohort study of 1009 episodes with community-acquired bacterial meningitis, we analyzed whether genetic variations in CFH influenced FH cerebrospinal fluid levels and/or disease severity. Subsequently, we analyzed the role of FH in our pneumococcal meningitis mouse model using FH knock-out (Cfh-/-) mice and wild-type (wt) mice. Finally, we tested whether adjuvant treatment with human FH (hFH) improved outcome in a randomized investigator blinded trial in a pneumococcal meningitis mouse model. RESULTS: We found the major allele (G) of single nucleotide polymorphism in CFH (rs6677604) to be associated with low FH cerebrospinal fluid concentration and increased mortality. In patients and mice with bacterial meningitis, FH concentrations were elevated during disease and Cfh-/- mice with pneumococcal meningitis had increased mortality compared to wild-type mice due to C3 depletion. Adjuvant treatment of wild-type mice with purified human FH led to complement inhibition but also increased bacterial outgrowth which resulted in similar disease outcomes. CONCLUSION: Low FH levels contribute to mortality in pneumococcal meningitis but adjuvant treatment with FH at a clinically relevant time point is not beneficial.

Histopathology of Listeria Meningitis.

Listeria monocytogenes meningitis is the third most common cause of bacterial meningitis in adults and has high mortality and morbidity rates. We describe the clinical course and score brain pathology of 5 patients who died of listeria meningitis. All patients were immunocompromised and ages ranged between 48 and 76 years. Three cases were confirmed by cerebrospinal fluid culture; one was confirmed by brain culture; and one diagnosis was based on a positive blood culture and neuropathological findings. Mild inflammation of meningeal arteries was found in 3 of 5 cases (60%). Moderate/severe ventriculitis was seen in 4 of 4 cases (100%), abscesses in 3 of 4 cases (75%), mild vascular inflammation in 4 of 5 cases (80%), mild/moderate hemorrhage in 2 of 4 cases (50%), mild/moderate thrombosis of meningeal artery in 3 of 5 cases (60%), and 1 case (25%) showed a moderate infarct. The inflammatory cells present in the meninges were characterized by a mix of monocytes, macrophages, and neutrophils and removal of apoptotic inflammatory cells by macrophages (efferocytosis). Gram stain showed intra- and extracellular presence of rod-shaped bacteria in 3 cases. Pathological examination was characterized by moderate to severe ventriculitis, abscesses and abundant efferocytosis which has been suggested to be exploited by L. monocytogenes for cell-to-cell spread.

Microglial Activation After Systemic Stimulation With Lipopolysaccharide and Escherichia coli.

Background: Microglial activation after systemic infection has been suggested to mediate sepsis-associated delirium. A systematic review of animal studies suggested distinct differences between microglial activation after systemic challenge with live bacteria and lipopolysaccharide (LPS). Here, we describe a mouse model of microglial activation after systemic challenge with live Escherichia coli (E. coli) and compare results with systemic challenge with LPS. Methods: Sixty mice were intraperitoneally injected with E. coli (1 × 104 colony-forming units) and sacrificed at 12, 20, 48, and 72 h after inoculation. For 48 and 72 h time points, mice were treated with ceftriaxone. Thirty mice were intraperitoneally injected with LPS (5 mg/kg) and sacrificed 3 and 48 h after inoculation; 48 control mice were intraperitoneally injected with isotonic saline. Microglial response was monitored by immunohistochemical staining with Iba-1 antibody and flow cytometry; and inflammatory response by mRNA expression of pro- and anti-inflammatory mediators. Results: Mice infected with live E. coli showed microglial activation 72 h post-inoculation, with increased cell number in cortex (p = 0.0002), hippocampus (p = 0.003), and thalamus (p = 0.0001), but not in the caudate nucleus/putamen (p = 0.33), as compared to controls. At 72 h, flow cytometry of microglia from E. coli infected mice showed increased cell size (p = 0.03) and CD45 expression (p = 0.03), but no increase in CD11b expression, and no differences in brain mRNA expression of inflammatory mediators as compared to controls. In mice with systemic LPS stimulation, microglial cells were morphologically activated at the 48 h time point with increased cell numbers in cortex (p = 0.002), hippocampus (p = 0.0003), thalamus (p = 0.007), and caudate nucleus/putamen (p < 0.0001), as compared to controls. At 48 h, flow cytometry of microglia from LPS stimulated mice showed increased cell size (p = 0.03), CD45 (p = 0.03), and CD11b (p = 0.04) expression. Brain mRNA expression of TNF-α (p = 0.02), IL-1ß (p = 0.02), and MCP-1 (p = 0.03) were increased as compared to controls. Interpretation: Systemic challenge with live E. coli causes a neuro-inflammatory response, but this response occurs at a later time point and is less vigorous as compared to LPS stimulation.The E. coli model mimics the clinical situation of infection associated delirium more closely than stimulation with supra-natural LPS.

Delayed cerebral thrombosis complicating pneumococcal meningitis: an autopsy study.

BACKGROUND: Delayed cerebral thrombosis (DCT) is a devastating cerebrovascular complication in patients with excellent initial recovery of pneumococcal meningitis. The aetiology is unknown, but direct bacterial invasion, activation of coagulation or post-infectious immunoglobulin deposition has been suggested. METHODS: We studied histopathology of 4 patients with pneumococcal meningitis complicated by DCT. Results were compared with 8 patients who died of pneumococcal meningitis without DCT and 3 non-meningitis control cases. Furthermore, we evaluated vascular immunoglobulin depositions (IgA, IgG and IgM) and the presence of pneumococcal capsules by immunofluorescence. RESULTS: Patients who died after pneumococcal meningitis showed inflammation in the meninges and blood vessels with extensive infarction and thrombosis. We did not observe gross differences between DCT and non-DCT patients, except that 2 of 4 DCT patients had a basilar artery aneurysm compared to none of the non-DCT patients. We observed high density of IgM and IgG deposition in meningitis cases as compared to controls, but no difference between DCT and non-DCT patients. Immunofluorescence staining of pneumococci demonstrated the presence of bacterial capsules in the meninges of all meningitis patients, even 35 days after the initiation of antibiotic treatment. CONCLUSION: The aetiology of DCT complicating pneumococcal meningitis seems to be of multifactorial aetiology and includes vascular inflammation, thromboembolism of large arteries and infectious intracranial aneurysms. Pneumococcal cell wall components can be observed for weeks after pneumococcal meningitis and may be a source of resurging inflammation after the initial immunosuppression by dexamethasone.

pIgR and PECAM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion.

Streptococcus pneumoniae is the main cause of bacterial meningitis, a life-threating disease with a high case fatality rate despite treatment with antibiotics. Pneumococci cause meningitis by invading the blood and penetrating the blood-brain barrier (BBB). Using stimulated emission depletion (STED) super-resolution microscopy of brain biopsies from patients who died of pneumococcal meningitis, we observe that pneumococci colocalize with the two BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1). We show that the major adhesin of the pneumococcal pilus-1, RrgA, binds both receptors, whereas the choline binding protein PspC binds, but to a lower extent, only pIgR. Using a bacteremia-derived meningitis model and mutant mice, as well as antibodies against the two receptors, we prevent pneumococcal entry into the brain and meningitis development. By adding antibodies to antibiotic (ceftriaxone)-treated mice, we further reduce the bacterial burden in the brain. Our data suggest that inhibition of pIgR and PECAM-1 has the potential to prevent pneumococcal meningitis.

C26:0-Carnitine Is a New Biomarker for X-Linked Adrenoleukodystrophy in Mice and Man.

X-linked adrenoleukodystrophy (ALD), a progressive neurodegenerative disease, is caused by mutations in ABCD1 and characterized by very-long-chain fatty acids (VLCFA) accumulation. Virtually all males develop progressive myelopathy (AMN). A subset of patients, however, develops a fatal cerebral demyelinating disease (cerebral ALD). Hematopoietic stem cell transplantation is curative for cerebral ALD provided the procedure is performed in an early stage of the disease. Unfortunately, this narrow therapeutic window is often missed. Therefore, an increasing number of newborn screening programs are including ALD. To identify new biomarkers for ALD, we developed an Abcd1 knockout mouse with enhanced VLCFA synthesis either ubiquitous or restricted to oligodendrocytes. Biochemical analysis revealed VLCFA accumulation in different lipid classes and acylcarnitines. Both C26:0-lysoPC and C26:0-carnitine were highly elevated in brain, spinal cord, but also in bloodspots. We extended the analysis to patients and confirmed that C26:0-carnitine is also elevated in bloodspots from ALD patients. We anticipate that validation of C26:0-carnitine for the diagnosis of ALD in newborn bloodspots may lead to a faster inclusion of ALD in newborn screening programs in countries that already screen for other inborn errors of metabolism.

Pneumococcal meningitis: clinical-pathological correlations (MeninGene-Path).

Pneumococcal meningitis is associated with substantial mortality and morbidity. We systematically assessed brain histopathology of 31 patients who died of pneumococcal meningitis from a nationwide study (median age 67 years; 21 (67 %) were male) using a pathology score including inflammation and vascular damage. Of the 27 patients with known time from the admission to death, 14 patients died within 7 days of admission and 13 after 7 days of admission. Eleven of 25 (44 %) patients had been treated with adjunctive dexamethasone therapy. Observed pathological processes were inflammation of medium-large arteries in 30 brains (97 %), cerebral haemorrhage in 24 (77 %), cerebritis in 24 (77 %), thrombosis in 21 (68 %), infarction in 19 (61 %) and ventriculitis in 19 (of 28 cases, 68 %). Inflammation of medium-large arteries led to obstruction of the vascular lumen in 14 (of 31 cases, 45 %). Vascular inflammation was associated with infarction and thrombosis of brain parenchymal vessels. Hippocampal dentate gyrus apoptosis between patients treated with and without dexamethasone was similar (p = 0.66); however, dexamethasone treated patients had higher total pathology score than non-dexamethasone treated patients (p = 0.003). Our study shows that vascular damage is key in the process of brain damage in pneumococcal meningitis. Data and material of this study will be made open-access for translational research in pneumococcal meningitis (MeninGene-Path).