Douglas A. Drevets

Subpopulations of Mouse Blood Monocytes Differ in Maturation Stage and Inflammatory Response

Blood monocytes are well-characterized precursors for macrophages and dendritic cells. Subsets of human monocytes with differential representation in various disease states are well known. In contrast, mouse monocyte subsets have been characterized minimally. In this study we identify three subpopulations of mouse monocytes that can be distinguished by differential expression of Ly-6C, CD43, CD11c, MBR, and CD62L. The subsets share the characteristics of extensive phagocytosis, similar expression of M-CSF receptor (CD115), and development into macrophages upon M-CSF stimulation. By eliminating blood monocytes with dichloromethylene-bisphosphonate-loaded liposomes and monitoring their repopulation, we showed a developmental relationship between the subsets. Monocytes were maximally depleted 18 h after liposome application and subsequently reappeared in the circulation. These cells were exclusively of the Ly-6Chigh subset, resembling bone marrow monocytes. Serial flow cytometric analyses of newly released Ly-6Chigh monocytes showed that Ly-6C expression on these cells was down-regulated while in circulation. Under inflammatory conditions elicited either by acute infection with Listeria monocytogenes or chronic infection with Leishmania major, there was a significant increase in immature Ly-6Chigh monocytes, resembling the inflammatory left shift of granulocytes. In addition, acute peritoneal inflammation recruited preferentially Ly-6Cmed-high monocytes. Taken together, these data identify distinct subpopulations of mouse blood monocytes that differ in maturation stage and capacity to become recruited to inflammatory sites.

Listeria monocytogenes : epidemiology, human disease, and mechanisms of brain invasion

Listeria monocytogenes is a facultative intracellular bacterium that has predilection for causing central nervous systemic infections in humans and domesticated animals. This pathogen can be found worldwide in the food supply and most L. monocytogenes infections are acquired through ingestion of contaminated food. The main clinical syndromes caused by L. monocytogenes include febrile gastroenteritis, perinatal infection, and systemic infections marked by central nervous system infections with or without bacteremia. Experimental infection of mice has been used for over 50 years as a model system to study the pathogenesis of this organism including the mechanisms by which it invades the brain. Data from this model indicate that a specific subset of monocytes, distinguished in part by high expression of the Ly-6C antigen, become parasitized in the bone marrow and have a key role in transporting intracellular bacteria across the blood-brain barriers and into the central nervous system. This Minireview will summarize recent epidemiologic and clinical information regarding L. monocytogenes as a human pathogen and will discuss current in vitro and in vivo data relevant to the role of parasitized monocytes and the pathogenetic mechanisms that underlie its formidable ability to invade the central nervous system.

Invasion of the Central Nervous System by Intracellular Bacteria

SUMMARY Infection of the central nervous system (CNS) is a severe and frequently fatal event during the course of many diseases caused by microbes with predominantly intracellular life cycles. Examples of these include the facultative intracellular bacteria Listeria monocytogenes, Mycobacterium tuberculosis, and Brucella and Salmonella spp. and obligate intracellular microbes of the Rickettsiaceae family and Tropheryma whipplei. Unfortunately, the mechanisms used by intracellular bacterial pathogens to enter the CNS are less well known than those used by bacterial pathogens with an extracellular life cycle. The goal of this review is to elaborate on the means by which intracellular bacterial pathogens establish infection within the CNS. This review encompasses the clinical and pathological findings that pertain to the CNS infection in humans and includes experimental data from animal models that illuminate how these microbes enter the CNS. Recent experimental data showing that L. monocytogenes can invade the CNS by more than one mechanism make it a useful model for discussing the various routes for neuroinvasion used by intracellular bacterial pathogens.

The Ly-6Chigh Monocyte Subpopulation Transports Listeria monocytogenes into the Brain during Systemic Infection of Mice

Mononuclear phagocytes can be used by intracellular pathogens to disseminate throughout the host. In the bloodstream these cells are generically referred to as monocytes. However, blood monocytes are a heterogeneous population, and the exact identity of the leukocyte(s) relevant for microbial spreading is not known. Experiments reported in this study used Listeria monocytogenes-infected mice to establish the phenotype of parasitized blood leukocytes and to test their role in systemic dissemination of intracellular bacteria. More than 90% of the blood leukocytes that were associated with bacteria were CD11b+ mononuclear cells. Analysis of newly described monocyte subsets showed that most infected cells belonged to the Ly-6Chigh monocyte subset and that Ly-6Chigh and Ly-6Cneg-low monocytes harbored similar numbers of bacteria per cell. Interestingly, systemic infection with wild-type or ΔactA mutants of L. monocytogenes, both of which escape from phagosomes and replicate intracellularly, caused expansion of the Ly-6Chigh subset. In contrast, this was not evident after infection with Δhly mutants, which neither escape phagosomes nor replicate intracellularly. Importantly, when CD11b+ leukocytes were isolated from the brains of lethally infected mice, 88% of these cells were identified as Ly-6Chigh monocytes. Kinetic analysis showed a significant influx of Ly-6Chigh monocytes into the brain 2 days after systemic infection. This coincided with both bacterial invasion and up-regulation of brain macrophage chemoattractant protein-1 gene expression. These data indicate that the Ly-6Chigh monocyte subset transports L. monocytogenes into the brain and establish their role as Trojan horses in vivo.

Listeria monocytogenes-Infected Phagocytes Can Initiate Central Nervous System Infection in Mice

ABSTRACT Listeria monocytogenes-infected phagocytes are present in the bloodstream of experimentally infected mice, but whether they play a role in central nervous system (CNS) invasion is unclear. To test whether bacteria within infected leukocytes could establish CNS infection, experimentally infected mice were treated with gentamicin delivered by surgically implanted osmotic pumps. Bacterial inhibitory titers in serum and plasma ranged from 1:16 to 1:256, and essentially all viable bacteria in the bloodstream of treated mice were leukocyte associated. Nevertheless, CNS infection developed in gentamicin-treated animals infected intraperitoneally or by gastric lavage, suggesting that intracellular bacteria could be responsible for neuroinvasion. This was supported by data showing that 43.5% of bacteria found with blood leukocytes were intracellular and some colocalized with F-actin, indicating productive intracellular parasitism. Experiments using an L. monocytogenes strain containing a chromosomal actA-gfpuv-plcB transcriptional fusion showed that blood leukocytes were associated with intracellular and extracellularly bound green fluorescent protein-expressing (GFP+) bacteria. Treatment with gentamicin decreased the numbers of extracellularly bound GFP+ bacteria significantly but did not affect the numbers of intracellular GFP+ bacteria, suggesting that the latter were the result of intercellular spread of GFP+ bacteria to leukocytes. These data demonstrate that infected leukocytes and the intracellularL. monocytogenes harbored within them play key roles in neuroinvasion. Moreover, they suggest that phagocytes recruited to infected organs such as the liver or spleen are themselves parasitized by intercellular spread of L. monocytogenes and then reenter the bloodstream and contribute to the systemic dissemination of bacteria.

Listeria monocytogenes Strains Selected on Ciprofloxacin or the Disinfectant Benzalkonium Chloride Exhibit Reduced Susceptibility to Ciprofloxacin, Gentamicin, Benzalkonium Chloride, and Other Toxic Compounds

ABSTRACT Listeria monocytogenes is a leading agent for severe food-borne illness and death in the United States and other nations. Even though drug resistance has not yet threatened therapeutic interventions for listeriosis, selective pressure associated with exposure to antibiotics and disinfectants may result in reduced susceptibility to these agents. In this study, selection of several L. monocytogenes strains on either ciprofloxacin (2 μg/ml) or the quaternary ammonium disinfectant benzalkonium chloride (BC; 10 μg/ml) led to derivatives with increased MICs not only to these agents but also to several other toxic compounds, including gentamicin, the dye ethidium bromide, and the chemotherapeutic drug tetraphenylphosphonium chloride. The spectrum of compounds to which these derivatives exhibited reduced susceptibility was the same regardless of whether they were selected on ciprofloxacin or on BC. Inclusion of strains harboring the large plasmid pLM80 revealed that MICs to ciprofloxacin and gentamicin did not differ between the parental and plasmid-cured strains. However, ciprofloxacin-selected derivatives of pLM80-harboring strains had higher MICs than those derived from the plasmid-cured strains. Susceptibility to the antimicrobials was partially restored in the presence of the potent efflux inhibitor reserpine. Taken together, these data suggest that mutations in efflux systems are responsible for the multidrug resistance phenotype of strains selected on ciprofloxacin or BC.

Leukocyte-facilitated entry of intracellular pathogens into the central nervous system

Microbes use numerous strategies to invade the central nervous system. Leukocyte-facilitated entry is one such mechanism whereby intracellular pathogens establish infection by taking advantage of leukocyte trafficking to the central nervous system. Key components of this process include peripheral infection and activation of leukocytes, activation of cerebral endothelial cells with or without concomitant infection, and trafficking of infected leukocytes to and through the blood-brain or blood-cerebrospinal fluid barrier.

Minocycline and aspirin in the treatment of bipolar depression: a protocol for a proof-of-concept, randomised, double-blind, placebo-controlled, 2×2 clinical trial

Introduction New medication classes are needed to improve treatment effectiveness in the depressed phase of bipolar disorder (BD). Extant evidence suggests that BD is characterised by neural changes such as dendritic remodelling and glial and neuronal cell loss. These changes have been hypothesised to result from chronic inflammation. The principal aims of the proposed research is to evaluate the antidepressant efficacy in bipolar depression of minocycline, a drug with neuroprotective and immune-modulating properties, and of aspirin, at doses expected to selectively inhibit cyclooxygenase 1 (COX-1). Methods and analysis 120 outpatients between 18 and 55 years of age, who meet DSM-IV-TR criteria for BD (type I or II) and for a current major depressive episode will be recruited to take part in a randomised, double-blind, placebo-controlled, parallel-group, proof-of-concept clinical trial following a 2×2 design. As adjuncts to existing treatment, subjects will be randomised to receive one of the four treatment combinations: placebo-minocycline plus placebo-aspirin, active-minocycline plus placebo-aspirin, placebo-minocycline plus active-aspirin or active-minocycline plus active-aspirin. The dose of minocycline and aspirin is 100 mg twice daily and 81 mg twice daily, respectively. Antidepressant response will be evaluated by assessing changes in the Montgomery–Asberg Depression Rating Scale scores between baseline and the end of the 6-week trial. As secondary outcome measures, the anti-inflammatory effects of minocycline and aspirin will be tested by measuring pre-treatment and post-treatment levels of C reactive protein and inflammatory cytokines. Ethics and dissemination Minocycline has been widely used as an antibiotic in doses up to 400 mg/day. Low-dose aspirin has been safely used on a worldwide scale for its role as an antithrombotic and thrombolytic. The study progress will be overseen by a Data, Safety and Monitoring Board, which will meet once every 6 months. Results of the study will be published in peer-reviewed publications. Trial registration number Clinical Trials.gov: NCT01429272.

Adverse outcomes associated with the use of drotrecogin alfa (activated) in patients with severe sepsis and baseline bleeding precautions.

Background:Key clinical trials involving drotrecogin alfa (activated) (or recombinant human activated protein C) excluded patients with specific baseline bleeding precautions. However, not all such precautions are considered contraindications to treatment with recombinant human activated protein C in current product labeling. Objective:To compare outcomes of patients receiving recombinant human activated protein C with or without baseline bleeding precautions as defined by the Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) trial. Design:Retrospective medical record review. Setting:Two tertiary care institutions: An academic medical center and an affiliated Veterans Affairs Medical Center. Patients:All patients receiving recombinant human activated protein C for treatment of sepsis. Interventions:Demographic information, characteristics associated with inclusion and exclusion criteria of the PROWESS trial, and 30-day postdischarge outcomes. Measurements and Main Results:Seventy-three patients received recombinant human activated protein C. Serious bleeding events occurred in 7 of 20 patients (35%) with any baseline bleeding precaution vs. only 2 of 53 patients (3.8%) without any bleeding precautions (p < 0.0001). More patients with a baseline bleeding precaution died compared with patients without any bleeding precautions (65% vs. 24.5%, p = 0.0015). Patients with a baseline bleeding precaution had a higher mean Acute Physiology and Chronic Health Evaluation II score (27.5 vs. 22.7, p = 0.015). Multivariate analysis demonstrated that the presence of a baseline bleeding precaution was the only independent variable associated with occurrence of serious bleeding events. The presence of a baseline bleeding precaution, increased Acute Physiology and Chronic Health Evaluation II score, and the presence of bloodstream infection were independent variables associated with mortality. Conclusions:Patients with severe sepsis who received recombinant human activated protein C with baseline bleeding precautions as defined by product labeling had significantly higher rates of both serious bleeding events and deaths compared with those without bleeding precautions. These data suggest that strict adherence to PROWESS trial exclusion criteria would further limit serious bleeding events associated with the use of recombinant human activated protein C.

Innate Responses to Systemic Infection by Intracellular Bacteria Trigger Recruitment of Ly-6Chigh Monocytes to the Brain

Blood borne Listeria monocytogenes enter the CNS via migration of parasitized Ly-6Chigh monocytes, but the signals that trigger this migration are not known. To understand more completely events leading to monocyte recruitment, experiments presented here combined microarray analysis of gene expression in the brains of experimentally infected mice with measurements of bacterial CFU and serum cytokines following i.v. infection with L. monocytogenes. At 24 and 48 h postinfection, the brain was sterile but there were significant changes in transcriptional activity related to serum proinflammatory cytokines. Real-time PCR confirmed mRNA up-regulation of genes related to IFN-γ, IL-1, and TNF-α, although IFN-γ itself was not up-regulated in the brain. Infection with Δacta, but not Δhly mutants, increased serum concentrations of IFN-γ, IL-6, and to a lesser extent TNF-α. The brain was not infected but there was widespread mRNA up-regulation in it and an influx of Ly-6Chigh monocytes in Δacta-infected mice. Moreover, ΔactA-infected IFN-γ−/− mice had no brain influx of Ly-6Chigh monocytes despite normal monocyte trafficking from bone marrow to blood and spleen. Additionally, IFN-γ−/− mice showed diminished mRNA expression for monocyte-attracting chemokines, and significantly less CXCL9 and CXCL10 protein in the brain compared with normal mice. These data demonstrate that monocyte recruitment to the brain is independent of bacterial invasion of the CNS and is triggered by proinflammatory cytokines, in particular IFN-γ, produced by the innate immune response to intracellular infection in peripheral organs.