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Updated: Jul 7, 2026
Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
Granulocyte-macrophage-colony-stimulating factor-dependent peritoneal macrophage responses determine survival in
Donn Spight1, Bruce Trapnell, Bin Zhao
1Department of Surgery, University of Cincinnati, Division of Pulmonary Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio, USA.
Abstract:
Granulocyte-macrophage-colony-stimulating factor (GM-CSF) plays a critical role in innate immunity by stimulating the differentiation of tissue macrophages via the transcription factor PU.1. Previous studies showed that GMCSF-deficient(GM-CSF-/-) mice had susceptibility to and impaired clearance of group B streptococcal bacteria by macrophages. For these studies, we hypothesized that GM-CSF-/- mice have increased susceptibility to peritonitis caused by immune dysfunction of peritoneal macrophages. We examined the role of peritoneal macrophages in pathogen clearance, cytokine responses, and survival in a murine cecal ligation and puncture (CLP) model of peritonitis/sepsis. Surprisingly, CLP minimally affected survival in GM-CSF-/- mice while markedly reducing survival in wild-type mice. This was not explained by differences in the composition of microbial flora, rates of bacterial peritonitis, or sepsis, all of which were similar in GM-CSF-/- and wild-type mice. However, survival correlated with peritoneal and serum TNF-alpha and IL-6 levels that were significantly lower in GM-CSF-/- than in control mice. After peritoneal LPS instillation, GM-CSF-/- mice also had improved survival and reduced TNF-alpha and IL-6 responses. In vitro studies demonstrated reduced secretion of TNF-alpha and IL-6 by peritoneal macrophages isolated from sham GM-CSF-/- mice as compared with macrophages from sham control mice. Peritoneal instillation of GM-CSF-/-/PU.1+ macrophages, but not GM-CSF-/-/PU.1+ macrophages into GM-CSF-/- mice conferred susceptibility to death after CLP or peritoneal LPS exposure. These results demonstrate that GM-CSFY/PU.1-dependent peritoneal macrophage responses are a critical determinant of survival after experimentally induced peritonitis/sepsis or exposure to LPS and have implications for therapies to treat such infections.
Insights
Granulocyte-macrophage-colony-stimulating factor (GM-CSF) deficiency surprisingly improved survival in a sepsis model, linked to lower inflammatory cytokine responses from peritoneal macrophages. This highlights a novel role for GM-CSF in sepsis pathogenesis.
Area of Science:
- Immunology
- Infectious Disease
- Cell Biology
Background:
- Granulocyte-macrophage-colony-stimulating factor (GM-CSF) is crucial for innate immunity and macrophage differentiation via PU.1.
- GM-CSF-deficient mice previously showed impaired bacterial clearance and susceptibility to infection.
- The role of GM-CSF in peritonitis and sepsis, particularly concerning peritoneal macrophages, was investigated.
Purpose of the Study:
- To investigate the role of GM-CSF and peritoneal macrophages in survival during experimentally induced peritonitis/sepsis.
- To examine the impact of GM-CSF deficiency on pathogen clearance, cytokine responses, and survival in a murine cecal ligation and puncture (CLP) model.
- To elucidate the mechanisms underlying altered survival in GM-CSF-deficient mice during sepsis.
Main Methods:
- Murine cecal ligation and puncture (CLP) model to induce peritonitis/sepsis.
- Analysis of survival rates, bacterial peritonitis, and microbial flora composition.
- Measurement of peritoneal and serum cytokine levels (TNF-alpha, IL-6).
- Peritoneal lipopolysaccharide (LPS) instillation studies.
- In vitro studies using peritoneal macrophages from GM-CSF-deficient and wild-type mice.
- Bone marrow chimera studies involving GM-CSF-deficient/PU.1+ and GM-CSF-deficient/PU.1- macrophages.
Main Results:
- Surprisingly, GM-CSF-deficient mice exhibited significantly improved survival after CLP compared to wild-type mice.
- Survival in GM-CSF-deficient mice correlated with lower peritoneal and serum levels of TNF-alpha and IL-6.
- GM-CSF-deficient mice showed improved survival and reduced inflammatory responses after LPS instillation.
- Peritoneal macrophages from GM-CSF-deficient mice secreted lower levels of TNF-alpha and IL-6 in vitro.
- Restoration of GM-CSF-dependent PU.1+ peritoneal macrophages into GM-CSF-deficient mice conferred susceptibility to death.
Conclusions:
- GM-CSF and its downstream transcription factor PU.1-dependent peritoneal macrophage responses are critical determinants of survival in experimental peritonitis/sepsis.
- Dysfunctional GM-CSF/PU.1-mediated macrophage responses, characterized by reduced pro-inflammatory cytokine production, confer resistance to sepsis in mice.
- These findings have significant implications for developing novel therapeutic strategies for sepsis and related inflammatory conditions.
