A Pilot Study to Investigate Peripheral Low-Level Chronic LPS Injection as a Model of Neutrophil Activation in the Periphery and Brain in Mice.

Lipopolysaccharide-induced (LPS) inflammation is used as model to understand the role of inflammation in brain diseases. However, no studies have assessed the ability of peripheral low-level chronic LPS to induce neutrophil activation in the periphery and brain. Subclinical levels of LPS were injected intraperitoneally into mice to investigate its impacts on neutrophil frequency and activation. Neutrophil activation, as measured by CD11b expression, was higher in LPS-injected mice compared to saline-injected mice after 4 weeks but not 8 weeks of injections. Neutrophil frequency and activation increased in the periphery 4-12 h and 4-8 h after the fourth and final injection, respectively. Increased levels of G-CSF, TNFa, IL-6, and CXCL2 were observed in the plasma along with increased neutrophil elastase, a marker of neutrophil extracellular traps, peaking 4 h following the final injection. Neutrophil activation was increased in the brain of LPS-injected mice when compared to saline-injected mice 4-8 h after the final injection. These results indicate that subclinical levels of peripheral LPS induces neutrophil activation in the periphery and brain. This model of chronic low-level systemic inflammation could be used to understand how neutrophils may act as mediators of the periphery-brain axis of inflammation with age and/or in mouse models of neurodegenerative or neuroinflammatory disease.

Peripheral Low Level Chronic LPS Injection as a Model of Neutrophil Activation in the Periphery and Brain in Mice.

Lipopolysaccharide-induced (LPS) inflammation is used as model to understand the role of inflammation in brain diseases. However, no studies have assessed the ability of peripheral low-level chronic LPS to induce neutrophil activation in the brain. Subclinical levels of LPS were injected intraperitoneally into mice to investigate impacts on neutrophil frequency and activation. Neutrophil activation, as measured by CD11b expression, peaked in the periphery after 4 weeks of weekly injections. Neutrophil frequency and activation increased in the periphery 4-12 hours and 4-8 hours after the fourth and final injection, respectively. Increased levels of G-CSF, TNFa, IL-6, and CXCL2 were observed in the plasma along with increased neutrophil elastase, a marker of neutrophil extracellular traps, peaking 4 hours following the final injection. Neutrophils and neutrophil activation were increased in the brain of LPS injected mice when compared to saline-injected mice 4 hours and 4-8 hours after the final injection, respectively. These results indicate that subclinical levels of peripheral LPS induces neutrophil activation in the periphery and brain. This model of chronic low-level systemic inflammation could be used to understand how neutrophils may act as mediators of the periphery-brain axis of inflammation with age and/or in mouse models of neurodegenerative or neuroinflammatory disease.

Neutrophils as a potential therapeutic target in Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of dementia in the United States. Sporadic or late-onset AD remains incompletely understood, with age as the current greatest risk factor. Inflammation in general and neutrophils, a potent mediator of inflammation, have been shown to exacerbate AD associated dementia. This review explores the latest research on neutrophils in AD mouse models and in human cohort studies and discusses current gaps in research and needs for future studies. AD mouse models have shown neutrophil chemotactic migration towards amyloid beta plaques in the brain. Capillary blood flow stalling decreases blood perfusion to associated brain regions and mouse studies have demonstrated that anti-Ly6G antibodies lead to a decrease in capillary blood flow stalling and memory improvement. Several recent transcriptomic studies of blood and brain tissue from persons with AD have shown an upregulation in neutrophil-related genes, and studies have demonstrated neutrophil involvement in brain capillary adhesion, blood brain barrier breaching, myeloperoxidase release, and the propensity for neutrophil extracellular trap release in AD. Neutrophil-derived inflammation and regulation are a potential potent novel therapeutic target for AD progression. Future studies should further investigate neutrophil functionality in AD. In addition, other aspects of AD that may impact neutrophils including the microbiome and the APOE4 allele should be studied.

NMR Hydrophilic Metabolomic Analysis of Bacterial Resistance Pathways Using Multivalent Antimicrobials with Challenged and Unchallenged Wild Type and Mutated Gram-Positive Bacteria.

Multivalent membrane disruptors are a relatively new antimicrobial scaffold that are difficult for bacteria to develop resistance to and can act on both Gram-positive and Gram-negative bacteria. Proton Nuclear Magnetic Resonance (1H NMR) metabolomics is an important method for studying resistance development in bacteria, since this is both a quantitative and qualitative method to study and identify phenotypes by changes in metabolic pathways. In this project, the metabolic differences between wild type Bacillus cereus (B. cereus) samples and B. cereus that was mutated through 33 growth cycles in a nonlethal dose of a multivalent antimicrobial agent were identified. For additional comparison, samples for analysis of the wild type and mutated strains of B. cereus were prepared in both challenged and unchallenged conditions. A C16-DABCO (1,4-diazabicyclo-2,2,2-octane) and mannose functionalized poly(amidoamine) dendrimer (DABCOMD) were used as the multivalent quaternary ammonium antimicrobial for this hydrophilic metabolic analysis. Overall, the study reported here indicates that B. cereus likely change their peptidoglycan layer to protect themselves from the highly positively charged DABCOMD. This membrane fortification most likely leads to the slow growth curve of the mutated, and especially the challenged mutant samples. The association of these sample types with metabolites associated with energy expenditure is attributed to the increased energy required for the membrane fortifications to occur as well as to the decreased diffusion of nutrients across the mutated membrane.

NMR metabolomic analysis of bacterial resistance pathways using multivalent quaternary ammonium functionalized macromolecules.

INTRODUCTION: Multivalent antimicrobial dendrimers are an exciting new system that is being developed to address the growing problem of drug resistant bacteria. Nuclear Magnetic Resonance (NMR) metabolomics is a quantitative and reproducible method for the determination of bacterial response to environmental stressors and for visualization of perturbations to biochemical pathways. OBJECTIVES: NMR metabolomics is used to elucidate metabolite differences between wild type and antimicrobially mutated Escherichia coli (E. coli) samples. METHODS: Proton (1H) NMR hydrophilic metabolite analysis was conducted on samples of E. coli after 33 growth cycles of a minimum inhibitory challenge to E. coli by poly(amidoamine) dendrimers functionalized with mannose and with C16-DABCO quaternary ammonium endgroups and compared to the metabolic profile of wild type E. coli. RESULTS: The wild type and mutated E. coli samples were separated into distinct sample sets by hierarchical clustering, principal component analysis (PCA) and sparse partial least squares discriminate analysis (sPLS-DA). Metabolite components of membrane fortification and energy related pathways had a significant p value and fold change between the wild type and mutated E. coli. Amino acids commonly associated with membrane fortification from cationic antimicrobials, such as lysine, were found to have a higher concentration in the mutated E. coli than in the wild type E. coli. N-acetylglucosamine, a major component of peptidoglycan synthesis, was found to have a 25-fold higher concentration in the mid log phase of the mutated E. coli than in the mid log phase of the wild type. CONCLUSION: The metabolic profile suggests that E. coli change their peptidoglycan composition in order to garner protection from the highly positively charged and multivalent C16-DABCO and mannose functionalized dendrimer.