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Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
Differential tumor necrosis factor alpha expression and release from peritoneal mouse macrophages in vitro in
W Cui1, D C Morrison, R Silverstein
1Departments of Basic Medical Science, School of Medicine, University of Missouri at Kansas City, Kansas City, Missouri 64108, USA.
Abstract:
Viable Escherichia coli and Staphylococcus aureus bacteria elicited markedly different in vitro tumor necrosis factor alpha (TNF-alpha) responses when placed in coculture with peritoneal murine macrophages. These include quantitative differences in TNF-alpha mRNA expression and corresponding protein product secretion as well as kinetic differences in the profiles of the TNF-alpha responses. Further, lipopolysaccharide (from E. coli) is a major contributing factor to these differences, as revealed by comparative experiments with endotoxin-responsive (C3Heb/FeJ) and endotoxin-hyporesponsive (C3H/HeJ) macrophages. Nevertheless, the eventual overall magnitude of the TNF-alpha secretion of macrophages in response to S. aureus was at least equivalent to that observed with E. coli, while appearing at time periods hours later than the E. coli-elicited TNF-alpha response. Both the magnitude and kinetic profile of the TNF-alpha responses were found to be relatively independent of the rate of bacterial proliferation, at least to the extent that similar results were observed with both viable and paraformaldehyde-killed microbes. Nevertheless, S. aureus treated in culture with the carbapenem antibiotic imipenem manifests markedly altered profiles of TNF-alpha response, with the appearance of an early TNF-alpha peak not seen with viable organisms, a finding strikingly similar to that recently reported by our laboratory from in vivo studies (R. Silverstein, J. G. Wood, Q. Xue, M. Norimatsu, D. L. Horn, and D. C. Morrison, Infect. Immun. 68:2301-2308, 2000). In contrast, imipenem treatment of E. coli-cocultured macrophages does not significantly alter the observed TNF-alpha response either in vitro or in vivo. In conclusion, our data support the concept that the host inflammatory response of cultured mouse macrophages in response to viable gram-positive versus gram-negative microbes exhibits distinctive characteristics and that these distinctions are, under some conditions, altered on subsequent bacterial killing, depending on the mode of killing. Of potential importance, these distinctive in vitro TNF-alpha profiles faithfully reflect circulating levels of TNF-alpha in infected mice. These results suggest that coculture of peritoneal macrophages with viable versus antibiotic-killed bacteria and subsequent assessment of cytokine response (TNF-alpha) may be of value in clarifying, and ultimately controlling, related host inflammatory responses in septic patients.
Insights
Macrophages exhibit distinct tumor necrosis factor alpha (TNF-alpha) responses to gram-positive Staphylococcus aureus versus gram-negative Escherichia coli. Bacterial killing method, particularly with antibiotics, significantly alters these inflammatory profiles, mirroring in vivo responses.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Host inflammatory responses to bacterial infections are crucial for pathogen clearance but can lead to tissue damage.
- Tumor necrosis factor alpha (TNF-alpha) is a key cytokine mediating inflammatory responses.
- Distinct differences exist in how macrophages, a type of immune cell, respond to different bacterial species.
Purpose of the Study:
- To investigate and compare the in vitro inflammatory responses of murine macrophages to viable gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria.
- To determine the role of bacterial components like lipopolysaccharide (LPS) in modulating these responses.
- To examine how antibiotic treatment and bacterial killing affect macrophage-mediated TNF-alpha production.
Main Methods:
- Coculture of peritoneal murine macrophages with viable or killed Escherichia coli and Staphylococcus aureus.
- Quantification of TNF-alpha mRNA expression and protein secretion.
- Comparative experiments using endotoxin-responsive and endotoxin-hyporesponsive macrophage strains.
- Assessment of TNF-alpha responses following antibiotic treatment (imipenem) of bacteria.
Main Results:
- Macrophages showed significantly different quantitative and kinetic TNF-alpha responses to E. coli versus S. aureus.
- Lipopolysaccharide from E. coli contributed to differential responses, evident in experiments with varying macrophage responsiveness.
- Killing of S. aureus with imipenem induced an early TNF-alpha peak, unlike viable bacteria, and this mirrored in vivo findings.
- Imipenem treatment did not significantly alter the TNF-alpha response to E. coli.
Conclusions:
- Cultured mouse macrophages exhibit distinct inflammatory profiles in response to gram-positive versus gram-negative bacteria.
- Bacterial killing, especially via specific antibiotic treatments, can alter these inflammatory responses, sometimes mimicking in vivo observations.
- In vitro TNF-alpha profiles generated through this coculture model accurately reflect in vivo inflammatory responses, suggesting its utility for studying sepsis.
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