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.

PurposeRecent data show that the Ly-6Chi monocyte subpopulation transports intracellular Listeria monocytogenes (Lm) into the brains of systemically infected mice. However, it is not clear whether peripheral or CNS infection triggers these monocytes to enter the brain. These experiments sought to establish whether entry of Ly-6Chi monocytes into the brain required bacterial infection of that organ, and determine the extent to which systemic Lm infection altered global gene expression in the brain.MethodsC57BL/6 mice were infected i.v. with wild type (WT) Lm, or with ΔactA or Δhly Lm mutants that can (ΔactA) or cannot (Δhly) replicate intracellularly. The mice were euthanized, exsanguinated, and perfused. The brain was removed and analyzed for bacterial infection, monocyte influx, or gene expression. Lm infection was established by quantitative culture. Numbers of Ly-6Chi monocytes were measured by FACS analysis. Gene expression was measured using the BD Atlas 5K gene array and by real-time PCR. Cytokine concentrations in the serum were quantified using ELISA and cytometric bead array. Statistical and data analyses were performed with the Student's t-test and GeneSpring 5.0 software.ResultsSystemic infection with 105 CFU WT Lm and 107 CFU ΔactA Lm mutants induced a significant influx of Ly-6Chi monocytes into the brain concomitant with significant upregulation of MCP-1 gene expression in that organ. In contrast, infection with 107 CFU Δhly Lm mutants induced neither response. Kinetic analyses of gene expression in the brain and microbiological studies of WT Lm-infected mice demonstrated widespread gene upregulation at days 1 and 2 post-infection, even though the brains were sterile. Serum levels of IFN-γ, TNF-α, and IL-6 were significantly increased from day 1 post-infection.ConclusionsInitial recruitment of Ly-6Chi monocytes into the brain does not require brain infection and is not a generic response to systemic bacterial infection. However, it is necessary that infecting bacteria escape from phagosomes and replicate intracellularly. In addition to upregulation of MCP-1 gene expression, there is widespread gene upregulation in the brain before bacteria enter that organ. These data suggest that pro-inflammatory cytokines produced by the innate immune response to intracellular replication of Lm in peripheral organs modulate gene expression in the CNS, a key consequence of which is recruitment of Ly-6Chi monocytes, some of which also contain intracellular bacteria, into the brain.

However, the prevalence of nasal MRSA colonization (1%) was substantially lower than that observed at our urban public hospital in Atlanta, GA, where rates exceed 7%. Further studies are needed to assess the prevalence of MRSA in the Republic of Georgia and to determine why rates of MRSA colonization remain lower than those reported from the U.S. and a number of countries in Europe.