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Published on: July 28, 2011
Host-pathogen dynamics through targeted secretome analysis of stimulated macrophages
Mohd M Khan1, Marijke Koppenol-Raab2, Minna Kuriakose2
1Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Abstract:
The pattern recognition receptors (PRRs) facilitate an organism's first line of defense against interlopers and shape the overall innate immune response through sensing and sampling pathogen-associated molecular patterns (PAMPs). The Toll-like receptor (TLR) family is the prototypic PRR family. Upon recognition of PAMPs, TLRs promote MyD88 dependent and independent responses. Understanding how different PAMPs are recognized by their specific TLRs and how pathogen recognition initiates immune activation is an intense area of research. Previously, we have reported the discovery of the temporal changes in signaling cascades of macrophage proteome and secretome post-stimulation with three different PAMPs. To extend our global proteomics approach to targeted protein abundance quantification, we describe the macrophage secretome targeted proteomics assay. We chose three different pathogens that specifically stimulate diverse TLRs (TLR2, TLR4, and TLR7). Using a simple targeted proteomics approach, combining data-dependent acquisition with an inclusion list, an array of cytokines, chemokines, and transcription factors can be profiled for their secretome abundance. This strategy facilitates the profiling and validation of pathogen-specific temporal changes in the macrophage secretome.
Insights
This study introduces a targeted proteomics assay to quantify macrophage secretome changes after pathogen stimulation. This method profiles cytokines and chemokines, aiding the study of innate immune responses to specific pathogens.
Area of Science:
- Immunology
- Proteomics
- Cell Biology
Background:
- Pattern recognition receptors (PRRs) are crucial for innate immunity, detecting pathogen-associated molecular patterns (PAMPs).
- Toll-like receptors (TLRs) are key PRRs that initiate MyD88-dependent and independent signaling pathways upon PAMP recognition.
- Understanding pathogen recognition by specific TLRs and subsequent immune activation is vital for immunology research.
Purpose of the Study:
- To develop and describe a targeted proteomics assay for quantifying macrophage secretome changes.
- To profile temporal changes in secreted proteins following stimulation with distinct PAMPs via diverse TLRs.
- To validate pathogen-specific immune responses at the proteomic level.
Main Methods:
- Development of a macrophage secretome targeted proteomics assay.
- Utilizing data-dependent acquisition combined with an inclusion list strategy.
- Stimulation of macrophages with three different pathogens targeting TLR2, TLR4, and TLR7.
Main Results:
- The assay enables targeted quantification of cytokines, chemokines, and transcription factors in the macrophage secretome.
- Demonstrated the ability to profile pathogen-specific temporal changes in secreted proteins.
- Validated the proteomic approach for studying innate immune responses.
Conclusions:
- The described targeted proteomics strategy is effective for profiling pathogen-specific temporal changes in the macrophage secretome.
- This method facilitates the validation of immune responses initiated by diverse TLR activation.
- Provides a valuable tool for in-depth analysis of innate immunity.
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