macrophage polarization regulates src-family kinase ...€¦ · antigen presentation...
TRANSCRIPT
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Macrophage polarization regulates Src-family kinase
transcription
Emily EwanFreedman Lab
NIH T35 Medical Student Summer Research Program in Infection and Immunity
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Wynn et al. (2013). Nature, 496, 445–455.
Antimicrobial activity
Phagocytosis
Cytotoxicity
Antigen presentation
Inflammation
Macrophages exhibit a diverse array of functions
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Wynn et al. (2013). Nature, 496, 445–455.
Antimicrobial activity
Angiogenesis
PhagocytosisImmune
regulation
Cytotoxicity
Tissue repair & remodeling
Tissue development
Antigen presentation
Inflammation
Macrophages exhibit a diverse array of functions
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Malyshev & Malyshev. (2015). Biomed Research International, 2015, 341308. Mantovani et al. (2004). Trends in Immunology, 25(12), 677–686.
Macrophage polarization as a spectrum
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Malyshev & Malyshev. (2015). Biomed Research International, 2015, 341308. Mantovani et al. (2004). Trends in Immunology, 25(12), 677–686.
Anti-inflammatory‘M2-like’
IFNγ/LPS IL-4/IL-13
STAT6IL-10 production
Pro-inflammatory‘M1-like’
NF-κB & STAT1 IL-12 and TNFα production
M1/M2 Polarization Paradigm
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Immune responsePathogen elimination
ITAM-associated receptors are important for macrophage anti-microbial signaling
ITAM-associated receptors like FcγR and Dectin-1
Figure adapted from Tanya Freedman
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Figure adapted from Tanya Freedman
ITAM-associated receptors like FcγR and Dectin-1
ITAM-associated receptor signaling through Src-family kinases
Immune responsePathogen elimination
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Figure adapted from Tanya Freedman
Macrophages express 3 endogenous SFKs
ITAM-associated receptors like FcγR and Dectin-1
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Figure adapted from Tanya FreedmanFreedman, T. S. et al. (2015). Elife, 4.
LynA plays an important role in macrophage anti-microbial signaling
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Freedman, T. S. et al. (2015). Elife, 4.Noubade et al. (2014). Nature, 509:235–239.
LynA protein expression is upregulated with inflammatory priming
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Pro-inflammatoryAnti-microbial
Antigen presentation
Anti-inflammatoryTissue repair
Immune regulation
Transcriptional upregulation Transcriptional downregulation
Hypotheses
IL-6GM-CSF
IFNγ LPS
TGF-β
IL-13IL-4
IL-10
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Pro-inflammatoryAnti-microbial
Antigen presentation
Anti-inflammatoryTissue repair
Immune regulation
Transcriptional upregulation Transcriptional downregulation
Murray, P. et al. (2014). Immunity, 41(1), 14-20.
Hypotheses
IL-6GM-CSF
IFNγ LPS
TGF-β
IL-13IL-4
IL-10
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Pro-inflammatoryAnti-microbial
Antigen presentation
Anti-inflammatoryTissue repair/remodeling
Immune regulation
Methods
IL-6GM-CSF
IFNγ LPS
TGF-β
IL-13IL-4
IL-10
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iNos: n=2 for IL-4 & IL-10/TGF-β, n=3 for all others; Arg1: n=3 for IFNγ, IL-4, & GM-CSF, n=1 for IL-10/TGF-β, n=2 for all others; includes Ben Brian’s data
iNos is upregulated with IFNγ/LPSArg1 is upregulated with IL-4/IL-13
Fold
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iNos: n=2 for IL-4 & IL-10/TGF-β, n=3 for all others; Arg1: n=3 for IFNγ, IL-4, & GM-CSF, n=1 for IL-10/TGF-β, n=2 for all others; includes Ben Brian’s data
Arg1 is upregulated with IL-10/TGF-βFo
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Arg1 is upregulated with GM-CSF and IL-6
iNos: n=2 for IL-4 & IL-10/TGF-β, n=3 for all others; Arg1: n=3 for IFNγ, IL-4, & GM-CSF, n=1 for IL-10/TGF-β, n=2 for all others; includes Ben Brian’s data
Fold
cha
nge
from
con
trol
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LynA is upregulated in IFNγ±LPS
†n=4; data includes Ben Brian’s experiments
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†n=4; ‡n=1; all other conditions n=3; data includes Ben Brian’s experiments
LynA is upregulated in IFNγ±LPS
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†n=1 for Hck; all other Hck conditions n=2; data includes Ben Brian’s experiments
Hck is also upregulated with IFNγ/LPS
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†n=1 for Fgr; all other Fgr conditions n=2; data includes Ben Brian’s experiments
Fgr is downregulated with IFNγ/LPS
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Conclusions
Pro-inflammatoryAnti-microbial
Antigen presentation
Anti-inflammatoryTissue repair
Immune regulation
IFNγ LPSIL-4
IL-10
IL-13TGF-β
IL-6GM-CSF
?
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Conclusions
Pro-inflammatoryAnti-microbial
Antigen presentation
Anti-inflammatoryTissue repair
Immune regulation
IFNγ LPS IL-10
TGF-β
IL-6GM-CSF
?
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ITIM-associated receptors like SIRPα
Figures adapted from Tanya Freedman and Ben BrianPeng, Q. et al. (2012). PLoS ONE, 7(6), e39967.
Priming macrophages with LPS àdecrease in Dok3 protein expression
Future Directions
c-Cbl
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Dok3 appears downregulated with all polarization conditions
n=1, 4 hour time point
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ITIM-associated receptors like SIRPα
Figures adapted from Tanya Freedman and Ben BrianKyo et al. (2003). Genes to Cells, 8, 825-836.
Future Directions
c-Cbl
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C-Cbl shows a trend of upregulation with a subset of anti-inflammatory conditions
n=2, 4 hour time point, Ben Brian’s data
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Future Directions
• Investigate Src-family kinase signaling and regulation in disease-associated macrophages– Cancer– Arthritis
Lewis, C. E. et al. (2016). Cancer Cell, 30(1), 18–25.
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Thank you!• Freedman Lab
– Ben Brian– Whitney Swanson– Tanya Freedman
• Schwertfeger Lab• Shimizu Lab• U of M Medical School &
T35 Program– Andrea Stewart– Dr. Mueller
Funding: T35 Medical Student Training Grant
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References• Wynn TA, Chawla A, Pollard JW. (2013). Macrophage biology in development, homeostasis
and disease. Nature, 496, 445–455. doi: 10.1038/nature12034.• Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A., & Locati, M. (2004). The
chemokine system in diverse forms of macrophage activation and polarization. Trends in Immunology, 25(12), 677–686. PMID: 15530839.
• Malyshev, I., & Malyshev, Y. (2015). Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage “Switch” Phenotype. BioMed Research International, 2015, 341308. http://doi.org/10.1155/2015/341308.
• Murray, P., Allen, J., Biswas, S., Gilroy, D., Goerdt, S., et al. (2014).Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity, 41(1), 14-20. doi:10.1016/j.immuni.2014.06.008.
• Freedman, T. S. et al. (2015). LynA regulates an inflammation-sensitive signaling checkpoint in Macrophages. Elife, 4. PMID: 26517880.
• Noubade R, Wong K, Ota N, Rutz S, Eidenschenk C, Valdez PA, Ding J, Peng I, Sebrell A, Caplazi P, DeVoss J, Soriano RH, Sai T, Lu R, Modrusan Z, Hackney J, Ouyang W. 2014. NRROS negatively regulates reactive oxygen species during host defence and autoimmunity. Nature 509:235–239. doi: 10.1038/nature13152.
• Peng, Q., O’Loughlin, J. L., & Humphrey, M. B. (2012). DOK3 Negatively Regulates LPS Responses and Endotoxin Tolerance. PLoS ONE, 7(6), e39967. http://doi.org/10.1371/journal.pone.0039967
• Kyo S, Sada K, Qu X, Maeno K, Miah SM, Kawauchi-Kamata K, Yamamura H. 2003. Negative regulation of Lyn protein-tyrosine kinase by c-Cbl ubiquitin-protein ligase in Fc epsilon RI-mediated mast cell activation. Genes to Cells 8:825–836. doi: 10.1046/j.1365-2443.2003.00679.x.
• Lewis, C. E., Harney, A. S., & Pollard, J. W. (2016). Cancer Cell, 30(1), 18–25.