Induction of VEGFA and Snail-1 by meningitic Escherichia coli mediates disruption of the blood-brain barrier

Abstract

Escherichia coli is the most common Gram-negative bacterium that possesses the ability to cause neonatal meningitis, which develops as circulating bacteria penetrate the blood-brain barrier (BBB). However, whether meningitic E. coli could induce disruption of the BBB and the underlying mechanisms are poorly understood. Our current work highlight for the first time the participation of VEGFA and Snail-1, as well as the potential mechanisms, in meningitic E. coli induced disruption of the BBB. Here, we characterized a meningitis-causing E. coli PCN033, and demonstrated that PCN033 invasion could increase the BBB permeability through downregulating and remodeling the tight junction proteins (TJ proteins). This process required the PCN033 infection-induced upregulation of VEGFA and Snail-1, which involves the activation of TLR2-MAPK-ERK1/2 signaling cascade. Moreover, production of proinflammatory cytokines and chemokines in response to infection also promoted the upregulation of VEGFA and Snail-1, therefore further mediating the BBB disruption. Our observations reported here directly support the involvement of VEGFA and Snail-1 in meningitic E. coli induced BBB disruption, and VEGFA and Snail-1 would therefore represent the essential host targets for future prevention of clinical E. coli meningitis.

Keywords: Snail-1; bacterial meningitis; blood-brain barrier; tight junctions; vascular endothelial growth factor A.

Figure 1. Screening and characterization of the meningitic E. coli isolate PCN033

(A–C) 30 ExPEC strains, including 7 strains from human patients, 3 strains from avian species, and 20 strains from pigs, were tested for their in vitro invasion of hBMEC. E. coli RS218 and HB101 were used as positive and negative controls, respectively. Results are presented as the relative invasion compared with that of RS218. (D) Brief background of the strains that chosen for the in vivo experiments in Figure E and F. (E–F) Survival in the blood (E) and invasion of the brain (F) were determined in 8 strains showing high invasion in vitro. Data are expressed as mean ± SD. (G) Typical neurological signs in mice receiving the challenge of meningitic E. coli PCN033, including overexcited, paddling, circling, trembling, and opisthotonus. (H–K) Mice were intravenously infected with PCN033 (n = 5), RS218 (n = 5), or HB101 (n = 5) at 1 × 10⁷ CFUs for 2, 4, and 6 h, at which time point the bacterial loads in the blood, brains, kidneys, and spleens were compared. Data are expressed as mean ± SD. (L) Brain histopathological changes in PCN033-challenged mice with neurological signs were examined by H&E staining. Panels a–d represent the control meninges from uninfected mice (black arrows). Panels e-l represent the brains from challenged mice, and red arrows indicate meninges disruption (e–h), hyperaemia (f, i), inflammatory cells accumulation (e–g, i, j), and embattled neurons (k, l). (M) Histopathological changes in brains from PCN033-challenged pigs. Panel a shows the normal meninges (black arrow). Panels b–f represent the typical pathological lesions with meninges thickening or separation (b–d), hyperaemia (b–f), and embattled neurons (f), as indicated by the red arrows.

Figure 2. Meningitic E. coli PCN033 enhanced the permeability of BBB via inducing downregulation and disorganization of the TJ proteins

(A–B) Mice were challenged by PCN033 until the appearance of different neurological signs, and NaF was injected intraperitoneally to allow diffusion. Uptake of NaF in the brain was analyzed by the fluorescence relative to that in the peripheral blood. (C) Evan's blue was injected to evaluate the integrity of the BBB. Because of the large molecular weight of Evan's blue by binding of serum albumin, only the increased permeability of the brain was observed in the moribund mice. (D–E) Brain lysates from the challenged mice were assessed via Western Blotting for the expression of TJ proteins, including ZO-1, β-catenin, Occludin, and Claudin-5. The β-actin was used as the loading control, and densitometry of the bands was performed to analyze the change of the protein expression upon infection. (F–I) Total RNA extracted from brains was analyzed by real-time PCR for the transcription of the TJ proteins. The β-actin gene was used as the internal reference for the brain RNAs. (J–K) Western blotting analysis of the TJ proteins in hBMEC in response to infection. β-actin was used as the loading control, and densitometry was performed to analyze the difference. (L–O) Real-time PCR analysis of the TJ proteins transcription in RNAs from infected hBMEC. GAPDH was used as the internal reference for the cellular RNAs in vitro. Analyzed data are presented as mean ± SD from three independent assays. (P–Q) Mice were challenged with or without PCN033 and brains from both groups of mice were analyzed for the integrity of vascular endothelium by IHC and IF. The TJ proteins ZO-1, β-catenin, Occludin, and Claudin-5 were selected as the markers reflecting the integrity of the vascular endothelium. In mock group, the TJ proteins were uniformly and closely arranged (Figure 2P, black arrows); in infected group, the arrangement of the TJ proteins became gapped, disrupted, and out-of-order (Figure 2P, red arrows). CD34 was specifically applied for labeling the vessel (Figure 2Q). Scale bar indicates 50 μm.

Figure 3. PCN033 induction of VEGFA mediated BBB permeability enhancement via downregulation of TJ proteins

(A) Real-time PCR analysis of the VEGFA transcription in hBMEC in response to PCN033 infection at the MOI of 10. GAPDH was used as the internal reference for the quantitation. (B) Determination of the secretory VEGFA in the hBMEC culture supernatant by the ELISA kit. (C–D) ELISA analysis of the VEGFA amount in serum and brain lysates from the challenged mice exhibiting different symptoms. (E) Contribution of the VEGFA to the permeability of the BBB. VEGFA was injected into the mice at increasing doses and the BBB permeability was evaluated by Evan's blue. Motesanib diphosphate (25 mg/kg) was intraperitoneally administrated 2 h before PCN033 challenge, and the BBB permeability was tested by Evan's blue method. (F–I) Expression of the TJ proteins (ZO-1, β-catenin, Occludin and Claudin-5) in hBMEC upon infection with or without VEGFR inhibition. Data are expressed as mean ± SD from three independent experiments.

Figure 4. PCN033-induced upregulation of Snail-1 mediated the decrease in TJ proteins

(A) Real-time PCR analysis of Snail-1 transcription in hBMEC in response to PCN033 challenge. GAPDH was used as the internal reference. (B) Snail-1 transcription was determined in total brain RNAs from infected mice exhibiting different signs. The transcription of β-actin was used as the internal reference. (C) PCN033-induced upregulation of Snail-1 in hBMEC was blocked by Snail-1 shRNA transfection, but not by the scrambled transfection. (D–G) Snail-1 knocking-down via shRNA partly offset the downregulation of TJ proteins by PCN033 infection compared with that in cells with scrambled transfection. Results are shown as mean ± SD from three independent assays.

Figure 5. TLR2-MAPK-ERK1/2 signaling cascade is required for the induction of VEGFA and Snail-1 by PCN033

(A) Phosphorylation of ERK1/2 along with PCN033 infection. The β-actin was detected as the loading control. (B) Densitometrical analysis of the ERK1/2 activation in hBMEC 2 h post-infection, compared with that in uninfected cells. Data are calculated as the ratio of phospho-ERK1/2 to total ERK1/2. (C) Effects of the MAPK signaling inhibitors on the PCN033-induced upregulation of VEGFA. U0126 (selective inhibitor of ERK1/2) and SB202190 (selective inhibitor of p38) could significantly decrease the PCN033-induced upregulation of VEGFA, while SP600125 (specific inhibitor of JNK) could not. (D) Effects of the MAPK signaling inhibitors on the PCN033-induced upregulation of Snail-1. Selective ERK1/2 inhibitor U0126 could completely block the PCN033-induced upregulation of Snail-1. (E–F) Snail-1 knocking-down via shRNA in hBMEC did not affect the induction of VEGFA by PCN033, while blocking VEGFA pathway significantly decreased the upregulation of Snail-1. (G–H) Effects of the VEGFR inhibitors on PCN033-induced activation of ERK1/2 and the densitometric analysis. (I–J) TLR2 agonist Pam3CSK4 induced the activation of ERK1/2 in a dose-dependent manner. (K–L) Pam3CSK4 dose-dependently induced the upregulation of Snail-1 and VEGFA in hBMEC. Results are expressed as mean ± SD from three independent assays.

Figure 6. PCN033-induced production of cytokines and chemokines conduced to upregulation of VEGFA and Snail-1

(A) PCN033 infection of the hBMEC induced the upregulation of multiple cytokines and chemokines in a time-dependent manner, including IL-6, IL-1β, TNF-α, MCP-1, MIP-2, and GRO-α. (B–C) Multiplex analysis of the cytokines and chemokines in the serum and brains from challenged mice along with time. Partial cytokines results are presented here, which show significantly increased generation compared with uninfected mice. (D) QuantiGene expression profiling of the cytokines in brain RNAs from challenged mice. The representative cytokines, IL-6, IL-1β, and TNF-α, showed the time-dependent increase along with infection. (E–F) Stimulation of the cytokines (IL-6, IL-1β or TNF-α) on hBMEC at 10 ng/mL led to the upregulation of VEGFA and Snail-1 in a time-dependent manner. Data are means ± SD of results from independent experiments.

Figure 7. Schematic presentation of the importance of VEGFA and Snail-1 in meningitic E. coli induced disruption of the BBB

Meningitic E. coli invasion of the BMECs triggers the activation of TLR2-MAPK-ERK1/2 signaling cascade, leading to the upregulation of VEGFA and Snail-1. On one hand, VEGFA secretion and action on its receptor VEGFR negatively regulate the transcription of TJ proteins; on the other hand, induction of Snail-1 could also directly negatively regulate the TJ proteins, both resulting in the disruption of the BBB. In addition, the infection-induced production of multiple cytokines and chemokines effectively conduced to the upregulation of VEGFA and Snail-1, further leading to the damage of the BBB integrity.