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Updated: Aug 5, 2025
Depletion and Reconstitution of Macrophages in Mice
Published on: August 1, 2012
MyD88 and not TRIF knockout is sufficient to abolish LPS-induced inflammatory responses in bone-derived macrophages
Marinaliz Reynoso1, Stuart Hobbs1, Alexander L Kolb1
1U.S. Army Research Institute of Environmental Medicine, Natick, MA, USA.
Abstract:
Macrophages play an important role in the response to infection and/or repair of injury in tissues. To examine the NF-κB pathway in response to an inflammatory stimulus, we used wild-type bone-marrow-derived macrophages (BMDMs) or BMDMs with knockout (KO) of myeloid differentiation primary response 88 (MyD88) and/or Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF) via CRISPR/Cas9. Following treatment of BMDMs with lipopolysaccharide (LPS) to induce an inflammatory response, translational signalling of NF-κB was quantified via immunoblot and cytokines were measured. Our findings reveal that MyD88 KO, but not TRIF KO, decreased LPS-induced NF-κB signalling, and 10% expression of basal MyD88 expression was sufficient to partially rescue the abolished inflammatory cytokine secretion observed upon MyD88 KO.
Insights
MyD88 is crucial for NF-κB signaling in macrophages during inflammation. Even partial MyD88 expression can restore inflammatory cytokine secretion after lipopolysaccharide stimulation.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Macrophages are key immune cells involved in tissue response to infection and injury.
- The Nuclear Factor-kappa B (NF-κB) pathway is central to inflammatory responses.
- Myeloid differentiation primary response 88 (MyD88) and Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF) are critical adaptors in Toll-like receptor signaling.
Purpose of the Study:
- To investigate the roles of MyD88 and TRIF in NF-κB pathway activation by lipopolysaccharide (LPS).
- To quantify the impact of MyD88 and TRIF knockout on inflammatory signaling and cytokine production.
Main Methods:
- Utilized wild-type and knockout (KO) bone-marrow-derived macrophages (BMDMs) lacking MyD88 and/or TRIF, generated using CRISPR/Cas9.
- Stimulated BMDMs with lipopolysaccharide (LPS) to induce inflammation.
- Quantified NF-κB signaling via immunoblotting and measured cytokine levels.
Main Results:
- MyD88 knockout significantly reduced LPS-induced NF-κB signaling, while TRIF knockout had no significant effect.
- Partial restoration (10% basal expression) of MyD88 was sufficient to partially rescue abolished inflammatory cytokine secretion in MyD88 KO cells.
Conclusions:
- MyD88 is the primary adaptor mediating NF-κB activation and subsequent inflammatory cytokine release in response to LPS in macrophages.
- TRIF plays a less significant role in this specific inflammatory context.
- MyD88 exhibits a dose-dependent effect on inflammatory responses, with low expression levels capable of partial functional rescue.
