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Proteomic profiling of the macrophage secretome upon Toll-like receptor stimulation

Thursday, September 17, 2015 — Poster Session II

12:00 p.m. – 1:30 p.m.
FAES Terrace


  • M Koppenol-Raab
  • V Sjoelund
  • B Dutta
  • Z Benet
  • I Fraser
  • A Nita-Lazar


Toll-like receptors (TLRs) are an essential component of the innate immune response and are among the first to detect invading pathogens. The intracellular signaling events that occur upon TLR stimulation have been well studied. However, proteins released into the extracellular space are important for the communication between cells during an immune response. Targeted studies have identified various cytokines among the factors released upon TLR activation, but comprehensive studies of the secretome are limited. We are using mass spectrometry-based proteomic methods to study the secretome of activated macrophages. RAW264.7 cells were stimulated with ligands to TLR4, TLR2 and TLR7 for 0, 6 and 24 hours, and the conditioned media was processed for proteomic analysis by mass spectrometry. We observe that TLR4 stimulation results in a more distinct response than that identified with the TLR2 and TLR7 treatments, which exhibit more overlap. Both TLR2 and TLR7 signaling are dependent on the adapter MyD88 and likely involve many of the same factors, but activation of TLR4 results in MyD88-dependent and MyD88-independent signaling. Thus we would expect to identify proteins that are unique to the TLR4 stimulation. We also observe significant down-regulation of proteins after 24 hours of TLR4 stimulation, suggesting that this pathway is shut off faster than the TLR2 and TLR7 responses. In addition, we are combining the secretome data with a proteomic analysis of the intracellular changes in response to TLR stimulation and comparing these protein-level changes with gene expression data to investigate the regulation of TLR signaling at multiple levels.

Category: Systems Biology