MyD88 and not TRIF knockout is sufficient to abolish LPS‐induced inflammatory responses in bone‐derived macrophages

• Published in: FEBS letters Vol. 597; no. 9; pp. 1225 - 1232
• Main Authors: Reynoso, Marinaliz, Hobbs, Stuart, Kolb, Alexander L., Matheny, Ronald W., Roberts, Brandon M.
• Format: Journal Article
• Language: English
• Published: England 01.05.2023
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Adaptor Proteins, Vesicular Transport - genetics; Adaptor Proteins, Vesicular Transport - metabolism; bonemarrow‐derived macrophages; CRISPR/Cas9; inflammation; interleukin‐10; Lipopolysaccharides - metabolism; Lipopolysaccharides - toxicity; Macrophages - metabolism; Myeloid Differentiation Factor 88 - genetics; Myeloid Differentiation Factor 88 - metabolism; NF-kappa B - genetics; NF-kappa B - metabolism; TLR4; Toll-Like Receptor 4 - genetics; Toll-Like Receptor 4 - metabolism; tumour necrosis factor; tumour necrosis factor; CRISPR/Cas9; inflammation; TLR4; interleukin-10; bonemarrow-derived macrophages
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1002/1873-3468.14616

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. To investigate the response of the NF‐κB pathway to inflammation, we used bone‐marrow‐derived macrophages with or without MyD88 knockout (KO) and/or TRIF KO using CRISPR/Cas9. MyD88 KO, but not TRIF KO, reduces LPS‐induced cytokine expression. Furthermore, CRISPR/Cas9 removal of MyD88 in TRIF KO cells decreased LPS‐induced TNFα and IL‐10 secretion but to a lesser extent than MyD88 KO cells.