Role of Vimentin and Alpha7 Nicotinic Acetylcholine Receptor of the Critical Targets in Meningitic Pathogenic Triad Among Blood-Brain Barrier Breakdown

Bacterial meningitis (BM) remains a global public health problem with high rates of mortality and morbidity. It is still elusive how blood-borne bacteria cause meningitis through modulating vimentin-α7nAChR signaling axis even though significant progress has been achieved. The theory proposed by our previous studies, pathogenic triad of BM, is that primary process is triggered with pathogenic bacteria invading the blood-brain barrier (BBB). The host cell signal transduction pathways are initiated subsequently which activate nuclear factor kappa B (NF-κB) signaling. It results secretion of proinflammatory cytokines or chemokines followed by the recruitment of leukocytes transmigration into the central nervous system that contributes to inflammation and injury. This triad is throughout the whole pathogenesis of BM. Here, we suggest that vimentin (Vim) and α7nAChR (A7R), two critical membrane receptors on brain microvascular endothelial cells (BMEC) constituting BBB, play a vital role in the pathogenic triad as key targets among BBB breakdown.

To explore the function of Vim and A7R in the triad, we employed mice of Vim^-/- (KO^Vim), A7R^-/- (KO^A7R) and wildtype (WT) to isolate BMEC, polymorphonuclear neutrophil (PMN) and construct animal model infected E. coli K1, a main BM pathogen. Bacterial invasion and PMN transmigration assays were performed to assess the effect of Vim in inflammatory. There were significant blocking effects of E. coli K1 invasion, PMN transmigration and NF-κB activation in KO^Vim BMEC in vitro and in vivo. Co-immunoprecipitation analysis shown that Vim could form a complex with IκB, NF-κB and tubulins in resting BMEC. The head domain of Vim was required for the complex formation. Two cytoskeletal components, Vim filaments and microtubules, contributed to the regulation of NF-κB. SiRNA knockdown studies demonstrated IKKα/β phosphorylation was completely abolished in BMEC lacking Vim and polypyrimidine tract-binding protein-associated splicing factor (PSF). Phosphorylation of ERK and nuclear translocation of NF-κB were entirely dependent on PSF. These findings imply that Vim and PSF cooperatively contribute to the meningitic pathogenic triad and the deficiency of Vim could protect mice from E. coli K1-induced meningitis.

Furthermore, bacterial invasion was significantly reduced in KO^A7Rin vitro and in vivo, with the same inhibiting effect and the reverse facilitating effect achieved by methyllycaconitine (MLA, A7R specific antagonist) and nicotine (NT, A7R specific agonist), respectively. The expression level of occludin and ZO-1, components of tight junction, was higher in the brain cortex of KO^A7R treated with NT compared to that in WT. Besides, proinflammatory cytokines (IL-1β, IL-6, TNF-α, MCP-1, MIP-1α) and adhesion molecules (CD44, ICAM-1) were significantly decreased in CSF of the KO^A7R mice with E. coli K1 meningitis. These indicate that A7R might function in the triad via enhancing BBB permeability and inflammatory. We further conducted a series of experiments of lipid raft fractionation, immunofluorescence microscopy and siRNA knockdown to explore the mechanism of A7R. Our results exhibit that lipid rafts play an essential role in A7R-mediated signaling. Next, in order to assess the effect of leukocytes transmigration across BBB driven by A7R, PMN transmigration assays were performed with integral KO^A7R and WT BMEC monolayer on Transwell filters. It was observed that the A7R deficiency of either BMEC or PMN resulted in a significant suppression of E. coli K1-induced PMN transmigration. Notably, it is of great interest to discover that memantine (MEM), an FDA-approved drug treating Alzheimer's disease, could very efficiently block BM caused by E. coli K1 in mice via a manner dependent on A7R. A set of assays carried out reveal that MEM serves this function through promoting the antibacterial function of neutrophil extracellular traps (NETs).

In conclusion, we elaborated that both Vim and A7R might play a detrimental role in the triad via destroying BBB. More importantly, the effect induced by A7R could be blocked by MEM. However, more investigations are still required to elucidate the underlying mechanism of interaction between Vim and A7R in the pathogenesis of BM. (Acknowledgements: J.Z. and X.G. contributed equally to this work; Corresponding author: Hong Cao, gzhcao@smu.edu.cn; National Natural Science Foundation of China, No. 81871198 to H.C.)

No relevant conflicts of interest to declare.