Macrophage polarization regulates Src-family kinase

transcription

Emily EwanFreedman Lab

NIH T35 Medical Student Summer Research Program in Infection and Immunity

Wynn et al. (2013). Nature, 496, 445–455.

Antimicrobial activity

Phagocytosis

Cytotoxicity

Antigen presentation

Inflammation

Macrophages exhibit a diverse array of functions

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

Malyshev & Malyshev. (2015). Biomed Research International, 2015, 341308. Mantovani et al. (2004). Trends in Immunology, 25(12), 677–686.

Macrophage polarization as a spectrum

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

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

Figure adapted from Tanya Freedman

ITAM-associated receptors like FcγR and Dectin-1

ITAM-associated receptor signaling through Src-family kinases

Immune responsePathogen elimination

Figure adapted from Tanya Freedman

Macrophages express 3 endogenous SFKs

ITAM-associated receptors like FcγR and Dectin-1

Figure adapted from Tanya FreedmanFreedman, T. S. et al. (2015). Elife, 4.

LynA plays an important role in macrophage anti-microbial signaling

Freedman, T. S. et al. (2015). Elife, 4.Noubade et al. (2014). Nature, 509:235–239.

LynA protein expression is upregulated with inflammatory priming

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

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

Pro-inflammatoryAnti-microbial

Antigen presentation

Anti-inflammatoryTissue repair/remodeling

Immune regulation

Methods

IL-6GM-CSF

IFNγ LPS

TGF-β

IL-13IL-4

IL-10

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

cha

nge

from

con

trol

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

ld c

hang

e fr

om c

ontr

ol

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

LynA is upregulated in IFNγ±LPS

†n=4; data includes Ben Brian’s experiments

†n=4; ‡n=1; all other conditions n=3; data includes Ben Brian’s experiments

LynA is upregulated in IFNγ±LPS

†n=1 for Hck; all other Hck conditions n=2; data includes Ben Brian’s experiments

Hck is also upregulated with IFNγ/LPS

†n=1 for Fgr; all other Fgr conditions n=2; data includes Ben Brian’s experiments

Fgr is downregulated with IFNγ/LPS

Conclusions

Pro-inflammatoryAnti-microbial

Antigen presentation

Anti-inflammatoryTissue repair

Immune regulation

IFNγ LPSIL-4

IL-10

IL-13TGF-β

IL-6GM-CSF

?

Conclusions

Pro-inflammatoryAnti-microbial

Antigen presentation

Anti-inflammatoryTissue repair

Immune regulation

IFNγ LPS IL-10

TGF-β

IL-6GM-CSF

?

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

Dok3 appears downregulated with all polarization conditions

n=1, 4 hour time point

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

C-Cbl shows a trend of upregulation with a subset of anti-inflammatory conditions

n=2, 4 hour time point, Ben Brian’s data

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.

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

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.