Study On The Effects And Mechanism Of Memantine Against Neonatal Spesis And Meningitis Through Alpha7 Cholinergic Pathway

BACKGROUND AND AIMSNeonatal sepsis and meningitis (NSM) remains a leading cause worldwide of mortality and morbidity in newborn infants despite the availability of antibiotics over the last several decades. In addition, about thirty percent of survivors can suffer permanent neurological sequelae, including seizures, deafness, hydrocephalus, cerebral palsy, and/or cognitive deficits, and so on. It mainly caused by bacteremia or sepsis and the pathogenic bacteria which causing NSM are constantly changing with the widespread use of antimicrobial agents. In Recent years, Staphylococcus epidermidis become the most common bacteria in hospital-acquired infection in the United States, but in the developing countries, E. coli is the most common gram-negative pathogen causing NSM, which is a leading cause of infection among neonates, particularly among those of very low birth weight. Fifty to sixty percent or even higher of the spesis with gram-negative pathogen are caused by E. coli and which can be isolated from the blood and cerebrospinal fluid (CSF) of NSM. E. coli is mainly from the birth canal or rectum which can translocate to vagina because of the special physical of pregnant women, leading to cause of infection in newborns when they go through the birth canal. Studies have shown that 20% of newborns with E. coli K1 colonization in the intestinal, among which most of their mother can be detected the same strain in the intestinal.0.5% of E. coli K1 that colonize neonatal intestine can be migrated across the BBB to the brain and cause meningitis.Recent studies suggest that there is an increasing incidence of early onset E. coli infections in low birth weight and very low birth weight neonates and a rising frequency of ampicillin-resistant E. coli infections in preterm infants. Widespread antibiotic use, particularly with the intrapartum prophylaxis use of antimicrobial agents, may result in a rising incidence of neonatal infections with antibiotic resistance, which is an ecological and evolutionary problem stemming from the response of bacteria to antibiotics. The ongoing antimicrobial resistance crisis will be certainly enhanced by antibiotic use, leading to the increasing global incidence of infectious diseases to which we have no known reliable antimicrobial agent.Despite the availability of highly bactericidal antibiotics over the last several decades, neonatal infections including spesis and meningitis remain a significant medical and economic problem. There are several major limitations inherent in the conventional antimicrobial drugs, which worsen the ecological and evolutionary problem. These medicines only target microbes based on the Manichaean view of the microbe-human host relationship. Almost all antimicrobial agents, regardless of spectrum of activity, kill both the good microbes, which may be beneficial to the host, as well as the bad germs. Focusing research on individual virulence genes and the important pathogens have been the traditional approach to human infectious diseases. Another limitation of this approach is due to the inability of many drugs to reach offending intracellular organisms and without regard for the host antimicrobial activities. Therefore, the generation of new anti-infective agents has emerged as an unmet need in the therapeutics of microbial infection including neonatal bacteremia and meningitis. Host-directed therapeutics against pathogens may provide more effective approaches to perturbing host pathways used by pathogens in various stages of their life cycle, namely, adhesion, invasion, and growth. Bacterial meningitis exhibits triad hallmark features (THFs):NF-κB activation, pathogen penetration and leukocyte transmigration across the blood-brain barrier (BBB), which consists mainly of brain microvascular endothelial cells (BMEC). The most challenging issue confronting neonatal bacterial meningitis is the lack of effective therapeutic interventions against the triad features of this disease.Our studies have shown that α7 nAChR, an essential regulator of inflammation, is critical for meningitic pathogen-induced triad features of neonatal sepsis and meningitis. a7 nAChR is abundantly expressed in hippocampus, the region most vulnerable to bacterial meningitis. Distinct regulatory mechanisms and functions have been revealed for activation of α7 nAChR, which is protective in adults but deleterious in neonates. Using the a7-deficient mouse cell cultures and animal model systems, we have demonstrated that α7 nAChR played a detrimental role to the host in penetration of E. coli and polymorphonuclear neutrophil (PMN) across the BBB and in neuronal inflammation. E. coli Kl invasion and PMN transmigration across the BBB were significantly reduced in α7-/- BMEC and α7-/- mice. Stimulation by nicotine was abolished in the α7-/- cells and animals. The same blocking effect was achieved by α7 antagonist methyllycaconitine (MLA). Neuronal inflammation, including secretion of proinflammatory cytokines and the inflammatory response in the hippocampus, was significantly reduced in the a7-deficient mice with E. coli meningitis. α7 nAChR-mediated calcium signaling in the wild-type brain endothelial cells was significantly enhanced upon exposure to nicotine and infection with the pathogen, while such cellular signaling was almost completely abolished in the α7-/-cells. These findings support the notion that a7 nAChR could serve as a unique drug target for therapeutic interventions against the triad features of neonatal sepsis and meningitis. In this report, using the drug repositioning approach and the in vitro/in vivo model systems of the BBB, we examined whether memantine, a FDA-approved drug for treatment of Alzheimer’s disease and also α7 antagonist, could be used as a host-directed antimicrobial agent against the triad features of neonatal bacterial meningitis. Indeed, our new drug repositioning studies have shown that memantine could very efficiently block E. coli-induced bacteremia and meningitic infections.Because of the immaturity of neonatal body barrier system including BBB, combined with the imperfect immune system, lacking of specific antibody, the meningitis become easily to occur in neonate. Most cases of neonatal E. coli meningitis develop as a result of hematogenous spread, and a critical step for the occurrence of E. coli meningitis is the circulating bacteria crossing blood-brain barrier (BBB), which mainly composed of brain microvascular endothelial cells (BMECs). To across the BBB successfully for E. coli, it needs to meet some certain prerequisites including a high degree of bacteremia, binding to and invasion of HBMEC, host cell cytoskeleton rearrangement and related signal pathway activation. Thus, suppression of pathogenic bacteria multiplying in blood circulation is the key to treatment of NSM. In this study, we have demonstrated that memantine can efficiency block bacteremia, resulting in the lower incidence of bacterial meningitis. So the following study will focus on the specific mechanisms of MEM in treatment of bacteremia with PMN.Neutrophil are important phagocytic cells, which is an important barrier for the non-specific immune system of host to resist the pathogenic microbial infection. Nowadays, it is generally accepted that killing mechanism of neutrophil is mainly through the intracellular mechanisms including phagocytosis, inactivated, digestion and so on. When agranulocytosis, the infection would be aggravated by the pathogen, moreover, the severe neutropenia become a leading cause of death in patients with bacterial infections. In this study, the results of animal experiments show that MEM has a significant effect on bacteremia, so that the incidence of bacterial meningitis is significantly reduced. In order to explore the specific mechanisms of MEM in bactericidal treatment and bacterial meningitis, we detect the effect of MEM on neutrophil phagocytosis. Whether MEM could enhance the ability of neutrophil phagocytosis and how MEM plays its bactericidal effect through α7 nAChR are the problems we would like to detect.In addition, the series studies of cell biology on neutrophil extracellular trap (neutrophil extracellular traps, NETs) provide us a novel strategy on our study of the mechanisms of memantine on NSM caused by meningitic E. coli. Except the phagocytosis, after neutrophils have been activated, phagocytic intracellular pathways in addition to sterilization, but it is activated, they will be proced to the programmed cell death and thus release nucleotide and bactericidal enzyme in the nucleus, forming a network (NETs) in the extracellular which can trap the invading bacteria and fungi. In this study, we would like to discuss Whether MEM can induce the formation of NETs while enhancing the ability of neutrophil phagocytosis, leading to better elaborate the specific mechanism of MEM in treatment of NSM and prone to lay a solid foundation for clinical application.Around the above clues, we would like to carry out the following studies:(1) Using the in vitro model to examine the drur targeting of MEM and assessed the antimicrobial activity of memantine in vitro; To test the blocking effects of MEM on a7 agonist nicotine-enhanced E. coli Kl invasion and to determine the numbers of surviving intracellular bacteria by the invasion assay or gentamicin survival assay after knocking-down the expression of CHRNA7 using siRNA; To determine whether blockage of NMDARs could affect intracellular survival of meningitic E. coli Kl in a manner similar to the inhibition of α7 nAChR, and perform comparative analysis of the effects of NMDAR and α7 nAChR inhibitors on bacterial intracellular survivals of HBMEC; (2) To determine the effects of MEM on tissue barriers; To test whether MEM was able to block E. coli infection across HBMEC with the bacterial invasion; Using the mouse model to examine the blocking effects of MEM on bacteremia, meningitis and BBB injury; (3) Using the in vitro model to examine the effects of MEM on inflammatory pathway with PMN transmigration assays; Using the mouse model to examine the blocking effects of MEM on inflammatory signaling and brain injury; (4) To detect the mechanism of MEM on bacterial killing. To test the effect of MEM on neutrophil phagocytosis assay and to perform analysis of the effects of α7 nAChR and bactericidal protein S100A9 on the bactericidal mechanism of MEM; To test whether MEM could induce the formation of neutrophil extracellular traps (NETs) by using the NETs kit and the immunofluorescence assay to further determine the specific bactericidal mechanism of MEM. (5) Using the in vitro and in vivo model to detect whether MEM was able to synergistically enhance the antibacterial activity of ampicillin in HBMEC infected with meningitic E. coli K1 and in neonatal mice with bacteremia and meningitis.METHODS AND RESULTS1. Comparative analysis of the effects of NMDAR and α7 nAChR inhibitors on a7 agonist nicotine-enhanced E. coli Kl invasion and its extracellular antimicrobial activity.1) To determine the drug targeting and if MEM could serve as a α7 antagonist to inhibit nicotine-enhanced pathogenicities of meningitic E. coli Kl, we examined blocking effects of this drug on pathogen penetration across HBMEC treated with and without nicotine. To mimic the concentrations of nicotine measured in the serum of human active and passive smokers, HBMEC were exposed to low doses of nicotine (10μM) for 48 hours, and then treated with different doses (1-50μM) of the α7 antagonist MEM. The cells were subjected to bacterial invasion assays. The result indicated that MEM was able to block E. coli invasion of HBMEC treated with nicotine in a dose-dependent manner.2) MEM has been shown to be the dual inhibitor of α7 nAChR and NMDARs, while it blocks a7 nAChR more potently than NMDARs in rat hippocampal neurons. To determine whether blockage of NMDARs could affect intracellular survival of meningitic E. coli Kl in a manner similar to the inhibition of α7 nAChR, we next performed comparative analysis of the effects of NMDAR and α7 nAChR inhibitors on bacterial intracellular survivals of HBMEC. The effects of MEM, NMDA (NMDA agonist) and two NMDAR antagonists, kynurenic acid (Kyn) and dextromethorphan (DM), were compared using the gentamicin survival assay. The results showed that DM and Kyn could not significantly block bacterial intracellular survivals of HBMEC and no dose-dependent effects were observed for these two drugs when compared to that of MEM. Furthermore, no significant stimulating effect was observed with the NMDAR agonist NMDA at 10μM that is the same dosage of the α7 agonist nicotine, which is able to significantly enhance E. coli Kl internalization of HBMEC. These findings demonstrate that MEM-mediated blockage of bacterial intracellular survivals mainly depends on α7 nAChR.3) To further determine whether α7 nAChR contribute to MEM-mediated blocking effects on nicotine-enhanced bacterial invasion, we knockdown the expression of α7 nAChR in HBMEC using siRNA. We perform the western blot assay to determine the knockdown efficiency of CHRNA7 after HBMEC tranfected with CHRNA7 siRNA and scrambled siRNA. After knockdown the expression of CHRNA7, HBMEC were exposed to low doses of nicotine (10μM) for 48 hours, and then treated with different doses (1-50μM) of the α7 antagonist MEM. The cells were subjected to bacterial invasion assays. The result indicated that the blocking effect of on E. coli invasion of HBMEC was significantly reduce after knockdown the expression of α7 nAChR in HBMEC. Taken together, these studies suggest that α7 nAChR contributes to MEM-mediated blocking effects on nicotine-enhanced bacterial invasion and PMN transmigration across HBMEC.4) Because our objective was to repurpose MEM as a drug that targets host rather than bacterial functions, we examined if this agent could act directly on E. coli Kl. We assessed the antimicrobial activity of MEM in vitro by growing bacteria in the presence of the drug. Bacteria were incubated with up to 100μM of MEM in BHI (Brain-heart infusion broth) for 1 to 8 hours at 37℃. The bacterial growth curves were determined by measuring the optical absorbance (OD600) at each time point. The growth rates of bacteria were compared in the presence of different concentrations of MEM at different time points. While this assay cannot distinguish between bacteriostatic and bactericidal activities of the drug, similar growth curves were obtained with or without MEM, regardless of concentrations or incubation periods. This result demonstrates that in the extracellular environment, MEM is unable to inhibit E. coli multiplication. Collectively, these data suggest that MEM can very efficiently block intracellular survival of E. coli Kl but has no extracellular antimicrobial activity.2. MEM was able to efficiently block bacterial intracellular survival in HBMEC and neonatal mice with bacteremia and meningitis.1) To examine whether MEM was able to block E. coli infection, we first determined the effects of this drug on bacterial internalization and survival in brain endothelial cells. HBMEC were infected with E44 and incubated with various concentrations of the drug before, during and after bacterial infection. The numbers of surviving intracellular bacteria were determined by the invasion assay or gentamicin survival assay as described in Materials and Methods. The data demonstrate that MEM could dose-dependently inhibit bacterial intracellular survival, no matter whether the drug was present in the systems before, during and after bacterial infection. These data suggest that this drug is a potential medication used to prevent and treat meningitic infection.2) To further determine the biological relevance of the in vitro assays, the efficacy of MEM on neonatal E. coli K1 meningitis was tested in the mouse model, as described in Methods and Materials. First, we investigated whether MEM could dose-dependently block bacteremia and meningitis in neonatal mice (7-9 day-old). Mice were treated with different doses (1-20mg/kg) of MEM during E44 infection (2×105 CFU). Our data show that this drug could dose-dependently block bacteremia and meningitis. MEM reduced median bacterial loads in blood and CSF by 2.31og10 to 7.71og10 CFU/ml in response to the drug treatment at doses of 1 to 20 mg/kg. The blocking effects in this range are statistically significant (P<0.05). Both bacteremia and meningitis are almost completely blocked by MEM at the dose of 20mg/ml. In the second experiment, wild-type neonatal (7-9 day-old) mice were divided into four groups. They were intraperitoneally injected with or without E44 (2×105 CFU) and treated with or without MEM (20mg/kg) during bacterial inoculation. We found that MEM was able to significantly block E. coli bacteremia (P<0.05) and bacterial entry into CSF (meningitis) (P<0.05). MEM could also significantly reduce the blood level of the BBB cellular biomarker cBMEC. These results suggest that MEM could decrease the host susceptibility to E. coli Kl infection (reduced bacteremia and meningitis), BBB injury (reduced cBMEC in blood).3. Blocking effects of MEM on PMN transmigration across HBMEC, inflammatory signaling and brain injury.1) To further determine the effects of MEM on inflammatory pathway, we examined blocking effects of this drug on PMN transmigration across HBMEC treated with and without nicotine. To mimic the concentrations of nicotine measured in the serum of human active and passive smokers, HBMEC were exposed to low doses of nicotine (10μM) for 48 hours, and then treated with different doses (1-50μM) of the α7 antagonist MEM. The cells were subjected to PMN transmigration assays. In order to exclude the possibility that the leukocyte migration elicited was due to destruction of HBMEC, the integrity of the monolayer was inspected by microscopy. MEM was able to significantly inhibit nicotine-enhanced PMN transmigration across the HBMEC monolayer in a dose-dependent manner. MEM-mediated blocking effects were observed upon treatment of either cell type alone or both, suggesting that a7 nAChR expression on both leukocytes and HBMEC are required for nicotine-enhanced PMN transmigration in vitro. These findings were consistent with the result of chemical blockage by the α7 antagonist MLA, suggesting that a7 nAChR on BMEC and PMN is required for leukocyte transmigration. Taken together, these studies suggest that α7 nAChR contributes to MEM-mediated blocking effects on nicotine-enhanced bacterial invasion and PMN transmigration across HBMEC.2) To further determine the effects of MEM on inflammatory signaling and brain injury, wild-type neonatal (7-9 day-old) mice were divided into four groups. They were intraperitoneally injected with or without E44 (2×105 CFU) and treated with or without MEM (20mg/kg) during bacterial inoculation. We found that MEM was able to significantly block E. coli bacteremia (P<0.05) and bacterial entry into CSF (meningitis) (P<0.05). MEM could also significantly reduce the magnitude of NF-κB activation (p65 in CSF) and the CSF concentration of MMP 9 when compared to the controls without drug treatment. These results suggest that MEM could decrease the CNS inflammatory response (reduced p65 and MMP 9 in CSF). The immunohistochemistry results showed that, as compared with the control group, MEM could significantly reduce the brain damage and aging in neonatal mice with NM.4. Study of the bactericidal mechanisms of MEM1) Our previous studies have shown that α7 nAChR act as an important role in blocking effects of MEM on α7 agonist nicotine-enhanced E. coli K1 invasion and PMN transmigration across HBMEC. Therefore, in order to further investigate the specific bactericidal mechanism of MEM and explore the interaction of α7 nAChR and bactericidal protein S100A9 in its bactericidal mechanisms, we first exam the bactericidal activitives of PMN and astrocytre cells, then we use siRNA to knockdown the expression of α7 nAChR and S100A9 in differentiation of HL60 cells (dHL60) which using 1.3% DMSO treatment for 5 days and western blotting was used to detect their knockdown efficency. The results showed that α7 nAChR and S100A9 gene expression were significantly reduced. Interestingly, when we successfully knockdown the expression of α7 nAChR, the expression of S100A9 would also be knockdown, which suggest that S100A9 protein was regulated by α7 nAChR. Studies have shown that, S100A9 protein has a bactericidal function. The results of neutrophils phagocytosis assay showed that the number of surviving E44 was significantly reduced after knockdown α7 nAChR, however, the bactericidal function of MEM was blocked, which indicated that α7 nAChR contributes to MEM-mediated blocking effects on bacterial survival.2) In order to explore how the α7 nAChR contribute to the MEM bactericidal mechanisms and understand the relationship between α7 nAChR and S100A9 on the process of neutrophils phagocytosis. After neutrophils incubation with E44 in the presence of MEM for 1 hour, α7 nAChR and S100A9 antibodies were incubated overnight, we then use immunofluorescence microscopy to observe the expression and interation between α7 nAChR and S100A9. Untreated neutrophils which used as a control demonstrated a homogeneous distribution of α7 nAChR and S100A9, after E44 stimulation, S100A9 was redistributed towards the cell margin and perinuclear region. A colocalization of S100A9 with clustered α7 nAChR was observed in neutrophils. The recruitment of α7 nAChR to the cell membrance was increased when compared with the entreated cells. However, the redistribution of α7 nAChR and S100A9 was greatly reduced with the MEM treatment, suggesting the release of S100A9 from neutrophils to carry out the bactericidal function after treatment with MEM.5. MEM induced the formation of NETs and was involved in trapping bacteria of neutrophils1) Studies have been shown that, in addition to the action of phagocytosis, neutrophil could form a neutrophil extracellular trap (NETs) to kill the pathogens. Our studies also demonstrated that MEM could enhance the ability of neutrophil phagocytosis. Therefore, we imagine that if MEM could induce the formation of NETs while involved in the bactericidal process. In the present study, we use the NETs kit and the immunofluorescence method to examine the effect of MEM neutrophil extracellular traps (NETs) formation. The result indicated that MEM could stimulate the formation of NETs, suggesting that MEM involved in the bacteria capture of NETs.2) We use the Immunofluorescence staining method to evaluate the formation of NETs after stimulated with MEM, E44 and PMA. Results showed that, after 4 h stimulation with MEM in vitro, a large number of extracellular reticular formation (NETs), the structure contains DNA, bactericidal protein S100A9 and bactericidal enzyme myeloperoxidase (MPO) as compared with the control group. Collectively, these data suggest that MEM could very efficiency induced the formation of NETs, leading to the release of DNA, S100A9 and MPO which is harmful to the pathegon.6. MEM was able to synergistically enhance the antibacterial activity of ampicillin in HBMEC infected with meningitic E44 and in neonatal mice with spesis and meningitis1) In order to further define the mechanisms of MEM-mediated intracellular blockage of E. coli K1 infection, we have examined in vitro and in vivo whether a combination of MEM and an antibiotic is superior to either medication alone in the treatment of bacterial infection. We determined the effects of MEM in conjunction with ampicillin (Amp), one of the most common antibiotics used for treatment of neonatal sepsis which treats Group B streptococcus and susceptible E. coli strains. HBMECs in the 24-well plates were infected with E44 for 1 h at 37℃ and then incubated with gentamicin (10mg/ml) for 1 h to eliminate extracellular bacteria. Under this condition gentamicin does not affect intracellular growth of E44 (data not shown). HBMECs were treated with Amp (5 to 100μg/ml) or MEM (1 to 15μM) alone, versus MEM in combination with Amp at the concentrations indicated. Intracellular survival of E44 was determined at 1 h. For these experiments, the concentrations of drugs chosen only partially inhibited bacterial growth. Combination treatment of MEM with Amp showed a stronger inhibitory effect on E44 infection. Determination of a synergistic, additive or antagonistic effect of MEM and Amp combination was performed according to the median effect principle using the CalcuSyn Software (Biosoft). The CI values for the combination treatment of MEM and Amp were less than 1, suggesting that the drug combination is highly synergistic. Together, these data suggest that the combination of MEM and Amp produced a synergistic reduction in the survival of meningitic E. coli Kl in HBMEC.2) Furthermore, the biological relevance of the in vitro study has been confirmed in the mouse model of NSM. The therapeutic efficacy of MEM and Amp alone and in conjunction was investigated in terms of reduction in the magnitude of bacteremia and the number of bacteria in CSF of neonatal mice infected with E44. The treatment was started after 6 h of bacterial inoculation and continued for 14 h. The adjunct therapy of 20mg MEM with 20mg Amp/kg body weight was found to be synergistic.STATISTICAL ANALYSISAll values are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed with Student’s t-test for comparison of two groups, and with ANOVA for multiple comparisons. In vivo experiments data were analyzed by Pearson Chi-Square test. Differences with P< 0.05 were considered to be statistically significant.CONCLUSIONMEM represents a promising host-directed antimicrobial agent that can be developed as a novel therapeutic intervention targeting host cells for the treatment of neonatal bacteremia and bacterial meningitis. Our data suggest that MEM may synergistically enhance the antimicrobial activity of the conventional antibiotics. We firstly predict that, on the early stage of BSM(bacteremia/sepsis), the bactericidal mechanism of MEM is mainly through inducing α7 nAChR which have a colocalization with bartericidal protein S100A9 after E44 stimulation. However, the redistribution of α7 nAChR and S100A9 was greatly reduced with the MEM treatment, and then bartericidal protein S100A9 was partly released from neutrophils to carry out the bactericidal function, together with the formation of NETs which including DNA, bartericidal protein S100A9 and and bactericidal enzyme MPO, leading to suppression the concentration of E44 in the blood that could block the bacteremia meningitis. While on the late stage og BSM(meningitis), We predict that, the bactericidal mechanism of MEM is mainly through blocking the α7 nAChR pathway to enhance the bartericidal activities of astrocyte cells, leading to reduce the inflammatory signaling and brain injury. Thus, this drug can efficiently block NSM at both the early and late stages of this disease. Because MEM targets host receptors such as α7 nAChR, not bacterial factors, it is more likely to reduce the risk of development of antimicrobial resistance compared to the conventional antibiotics.