HIF-1α is involved in blood–brain barrier dysfunction and paracellular migration of bacteria in pneumococcal meningitis

Bacterial meningitis is a deadly disease most commonly caused by Streptococcus pneumoniae, leading to severe neurological sequelae including cerebral edema, seizures, stroke, and mortality when untreated. Meningitis is initiated by the transfer of S. pneumoniae from blood to the brain across the blood–cerebrospinal fluid barrier or the blood–brain barrier (BBB). The underlying mechanisms are still poorly understood. Current treatment strategies include adjuvant dexamethasone for inflammation and cerebral edema, followed by antibiotics. The success of dexamethasone is however inconclusive, necessitating new therapies for controlling edema, the primary reason for neurological complications. Since we have previously shown a general activation of hypoxia inducible factor (HIF-1α) in bacterial infections, we hypothesized that HIF-1α, via induction of vascular endothelial growth factor (VEGF) is involved in transmigration of pathogens across the BBB. In human, murine meningitis brain samples, HIF-1α activation was observed by immunohistochemistry. S. pneumoniae infection in brain endothelial cells (EC) resulted in in vitro upregulation of HIF-1α/VEGF (Western blotting/qRT-PCR) associated with increased paracellular permeability (fluorometry, impedance measurements). This was supported by bacterial localization at cell–cell junctions in vitro and in vivo in brain ECs from mouse and humans (confocal, super-resolution, electron microscopy, live-cell imaging). Hematogenously infected mice showed increased permeability, S. pneumoniae deposition in the brain, along with upregulation of genes in the HIF-1α/VEGF pathway (RNA sequencing of brain microvessels). Inhibition of HIF-1α with echinomycin, siRNA in bEnd5 cells or using primary brain ECs from HIF-1α knock-out mice revealed reduced endothelial permeability and transmigration of S. pneumoniae. Therapeutic rescue using the HIF-1α inhibitor echinomycin resulted in increased survival and improvement of BBB function in S. pneumoniae-infected mice. We thus demonstrate paracellular migration of bacteria across BBB and a critical role for HIF-1α/VEGF therein and hence propose targeting this pathway to prevent BBB dysfunction and ensuing brain damage in infections. Electronic supplementary material The online version of this article (10.1007/s00401-020-02174-2) contains supplementary material, which is available to authorized users.

. HIF-1a staining of other human meningitis cases. Human brain paraffin specimen from different meningitis cases (indicated in the figure) were subjected to HIF-1a staining. Positive staining for HIF-1a was observed in different regions in all the specimens in vascular, neural and immune cells. The patient date of these cases is also included in table 1. Scale bar is 50 µm. Figure S4. Role of S. pneumoniae capsule in HIF1a induction and permeability. As the capsule of S. pneumoniae is critical for its virulence, we tested whether it is important for HIF1a induction and permeability. Using capsule deleted mutants of TIGR4 strain (∆cps), an induction of HIF-1a and VEGF similar to the wild-type strain was observed (top panel). Similar results were also obtained in dextran permeability experiments (bottom panel) suggesting no role of the S. pneumoniae capsule in HIF-1a induction and permeability. (Mean + SEM, N=2 experiments using bEnd5 cells for both qRT-PCR/permeability assays.

Figure S5. Loss of junction molecules upon S. pneumoniae infection in bEnd5 cells.
As S. pneumoniae infection of bEnd5 cells lead to permeability, Western blotting was performed to analyze junction molecules. VE-cadherin, an adherens junction marker was dramatically HIF-1a down regulated whereas clauin-5, a tight junction molecule was not altered. HIF-1a was upregulated as also shown in figure 2. Tubulin a, a house keeping protein served as the loading control. Representative Western blot from 2 experiments.

Figure S6. S. pneumoniae infection of pericytes but not astrocytes causes EC permeability.
As S. pneumoniae infection of brain ECs lead to permeability, the influence of other NVU cells in permeability of EC barrier upon S. pneumoniae infection was tested in vitro. Conditioned media from primary human brain vascular pericytes (PC) at 3 h post infection with TIGR4 strain lead to increase in permeability of bEnd5 cells in tendency but not using the media from primary mouse astrocytes (AC) (Mean + SEM, N=2 experiments).   S. pneumoniae infection of mice followed by isolation of microvessels and RNA sequencing was performed to study the mechanisms involved in transfer of S. pneumoniae across the BBB. Similar to KEGG and PANTHER analyses, KOBAS reactome analysis also indicated regulation of pathways related to cancer and inflammation. Pathways related to HIF-1a/VEGF signaling were also activated in the infected brain vessels when contrasted with healthy sham microvessels (green arrows) including regulation of cell adhesion molecules relevant to BBB permeability (red arrows). Genes significantly regulated per contrast (P-value < 0.05, Absolute (Log2FC) > 0.585) were included in the KOBAS enrichment.

Figure S10. HIF-1a induction and permeability upon S. pneumoniae infection of human brain ECs.
S. pneumoniae infection of human primary brain ECs also lead to induction of HIF-1a and VEGF (left graph). Similar to mouse brain ECs, increase in permeability was observed in tendency for human brain ECs to the higher molecular weight 70 kD dextran. Moreover, the effect appeared to be HIF-1a dependent (middle graph) as it was abrogated upon siRNA knockdown of HIF-1a in these cells, which was confirmed by qRT-PCR analysis (right graph).

Figure S11. Echinomycin rescues BBB function over vehicle closer to healthy controls.
S. pneumoniae infection of mice followed by echinomycin treatment leads to improved BBB phenotype compared to the vehicle treated mice. While (A) HIF-1a staining was decreased in echinomycin group, there was no change in (B) S.pneumoniae (Spn) staining. The expression of BBB functional markers (C, D) in echinomycin treated animals were closer to healthy controls indicating a therapeutic rescue when compared untreated vehicle group. The control healthy animals however were negative for Spn indicating the staining specificity (B, top right panel). Scale bar is 10 microns except top right panel where it is 2.5 microns. Control animal staining is representative of 6 animals.