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Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages
Published on: May 21, 2018
Differences in the sensitivity of classically and alternatively activated macrophages to TAK1 inhibitor-induced
Zsófia Varga1,2, Tamás Molnár1,2, Anett Mázló1,2,3
1Department of Immunology, Faculty of Medicine, University of Debrecen, 1 Egyetem Square, Debrecen, 4032, Hungary.
Abstract:
Controlling the balance of pro-inflammatory M1 versus anti-inflammatory M2 macrophages may have paramount therapeutic benefit in cardiovascular diseases, infections, cancer and chronic inflammation. The targeted depletion of different macrophage populations provides a therapeutic option to regulate macrophage-mediated functions. Macrophages are highly sensitive to necroptosis, a newly described regulated cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain like pseudokinase. Antagonists of inhibitors of apoptosis proteins (SMAC mimetics) block RIPK1 ubiquitination, while TGF-activated kinase 1 (TAK1) inhibitors prevent the phosphorylation of RIPK1, resulting in increased necroptosis. We compared the sensitivity of monocyte-derived human M1 and M2 cells to various apoptotic and necroptotic signals. The two cell types were equally sensitive to all investigated stimuli, but TAK1 inhibitor induced more intense necroptosis in M2 cells. Consequently, the treatment of co-cultured M1 and M2 cells with TAK1 inhibitor shifted the balance of the two populations toward M1 dominance. Blockage of either Aurora Kinase A or glycogen synthase kinase 3β, two newly described necroptosis inhibitors, increased the sensitivity of M1 cells to TAK1-inhibitor-induced cell death. Finally, we demonstrated that in vitro differentiated tumor-associated macrophages (TAM-like cells) were as highly sensitive to TAK1 inhibitor-induced necroptosis as M2 cells. Our results indicate that at least two different necroptotic pathways operate in macrophages and the targeted elimination of different macrophage populations by TAK1 inhibitor or SMAC mimetic may provide a therapeutic option to regulate the balance of inflammatory/anti-inflammatory macrophage functions.
Insights
Targeting macrophage necroptosis with TAK1 inhibitors can shift the balance from anti-inflammatory M2 to pro-inflammatory M1 macrophages, offering therapeutic potential for inflammatory diseases.
Area of Science:
- Immunology
- Cell Death Research
- Molecular Biology
Background:
- Macrophages play critical roles in inflammation, immunity, and disease.
- Balancing pro-inflammatory (M1) and anti-inflammatory (M2) macrophage populations is therapeutically important for various conditions.
- Necroptosis, a regulated form of cell death, is mediated by RIPK1, RIPK3, and MLKL.
Purpose of the Study:
- To investigate the differential sensitivity of M1 and M2 macrophages to necroptosis.
- To explore the potential of targeting necroptosis pathways for modulating macrophage polarization.
- To assess the efficacy of specific necroptosis inhibitors in shifting macrophage balance.
Main Methods:
- Comparison of monocyte-derived human M1 and M2 cell sensitivity to apoptotic and necroptotic stimuli.
- Treatment with TGF-activated kinase 1 (TAK1) inhibitors to induce necroptosis.
- Assessment of macrophage population shifts in co-cultured M1 and M2 cells.
- Evaluation of necroptosis inhibitors (Aurora Kinase A, GSK3β) and their effect on M1 cell sensitivity.
- Analysis of tumor-associated macrophage (TAM-like) sensitivity to TAK1 inhibitors.
Main Results:
- M1 and M2 macrophages exhibited similar sensitivity to most stimuli, but M2 cells were more sensitive to TAK1 inhibitor-induced necroptosis.
- TAK1 inhibitor treatment shifted co-cultured M1/M2 populations towards M1 dominance.
- Inhibiting Aurora Kinase A or GSK3β enhanced M1 cell sensitivity to TAK1 inhibitor-induced death.
- In vitro differentiated TAM-like cells showed high sensitivity to TAK1 inhibitor-induced necroptosis, similar to M2 cells.
Conclusions:
- Macrophages are sensitive to necroptosis, with distinct pathways potentially operating.
- Targeted necroptosis induction, particularly via TAK1 inhibition, can selectively eliminate M2 macrophages and promote an M1-dominant phenotype.
- This approach offers a therapeutic strategy for modulating macrophage balance in inflammatory and immune-related diseases.
- SMAC mimetics and TAK1 inhibitors represent potential tools for regulating macrophage populations.
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