Uptake and killing of Listeria monocytogenes by normal human peripheral blood granulocytes and monocytes as measured by flow cytometry and cell sorting

Abstract

Cellular components of innate immunity (NK cells, monocytes and granulocytes) play an important role in early resistance to Listeria monocytogenes in the mouse model. Minimally invasive methods of measuring the bacteriocidal capacity of these cells may be useful as a biomarker of susceptibility in humans. A technique was developed whereby the uptake and survival of L. monocytogenes could be measured in human granulocytes and monocytes using small volumes of peripheral blood. This method used flow cytometry to detect the presence of PKH-2-labeled bacteria within these cells. Survival of bacteria was determined by sorting of infected cells based on a combination of fluorescence and light scattering properties. Considerable variation in bacterial recovery was seen between normal volunteers. There was consistently greater survival of a fully virulent strain of L. monocytogenes within monocytes and granulocytes compared with an isogenic strain lacking the hemolysin, listeriolysin O, when measured at baseline. There was no evidence of longer-term bacterial survival or growth at 2 or 24 h. This technique may be useful for assessment of both host resistance and pathogen virulence.

Monocytes Are the Predominant Cell Type Associated with Listeria monocytogenes in the Gut, but They Do Not Serve as an Intracellular Growth Niche

“…This is in agreement with Raybourne et al who suggested that human blood monocytes could not support the growth of L. monocytogenes over time (48). It is possible that activity of the pore-forming toxin LLO is impaired in monocytes.…”

LIGHT enhances the bactericidal activity of human monocytes and neutrophils via HVEM

“…Killing of bacteria was measured as described previously [25,27]. Briefly, overnight cultures of L. monocytogenes (1ϫ10 7 cells/ml) and S. aureus (1ϫ10 7 cells/ml) were opsonized by incubation with 0.1% gelatin (w/v) and 10% (v/v) human AB serum in HBSS, where 1 ml containing 1 ϫ 10 7 opsonized bacteria was added to 1 ϫ 10 7 cells/ml in 100 l HBSS and incubated at 37°C for 3 min with continuous rotation to promote phagocytosis.…”

Proteogenomics in Aid of Host–Pathogen Interaction Studies: A Bacterial Perspective

“…Using microscopy, the number and the distribution or location of bacteria within host cells or cell-associated bacteria can be determined in a quantitative manner as, for example, shown by Malik-Kale et al [ 30 ] who made use of mCherry expressing Salmonella . By measuring mean fluorescence intensities, flow cytometry enables semi-quantitative analyses as demonstrated by Raybourne et al [ 31 ] who compared the intracellular L. monocytogenes content of human monocytes and granulocytes by pre-labeling bacteria with a lipophilic fluorescent dye. Isolation of infected cells using fluorescence-activated cell sorting (FACS) typically relies on the exogenous expression of fluorescent proteins, like green fluorescent protein (GFP), by pathogens [ 32 , 33 , 34 , 35 ].…”