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Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis
Published on: November 28, 2015
Resolving macrophage polarization through distinct Ca2+ entry channel that maintains intracellular signaling and
Viviane Nascimento Da Conceicao1, Yuyang Sun1, Karthik Ramachandran2
1Department of Periodontics, University of Texas Health San Antonio, San Antonio, TX 78229, USA.
Abstract:
Transformation of naive macrophages into classically (M1) or alternatively (M2) activated macrophages regulates the inflammatory response. Here, we identified that distinct Ca2+ entry channels determine the IFNγ-induced M1 or IL-4-induced M2 transition. Naive or M2 macrophages exhibit a robust Ca2+ entry that was dependent on Orai1 channels, whereas the M1 phenotype showed a non-selective TRPC1 current. Blockade of Ca2+ entry suppresses pNF-κB/pJNK/STAT1 or STAT6 signaling events and consequently lowers cytokine production that is essential for M1 or M2 functions. Of importance, LPS stimulation shifted M2 cells from Orai1 toward TRPC1-mediated Ca2+ entry and TRPC1-/- mice exhibited transcriptional changes that suppress pro-inflammatory cytokines. In contrast, Orai1-/- macrophages showed a decrease in anti-inflammatory cytokines and exhibited a suppression of mitochondrial oxygen consumption rate and inhibited mitochondrial shape transition specifically in the M2 cells. Finally, alterations in TRPC1 or Orai1 expression determine macrophage polarization suggesting a distinct role of Ca2+ channels in modulating macrophage transformation.
Insights
Distinct calcium (Ca2+) channels, Orai1 and TRPC1, dictate macrophage polarization into M1 or M2 states, influencing inflammatory responses and cytokine production. Their specific roles highlight new therapeutic targets for immune modulation.
Area of Science:
- Immunology
- Cell Biology
- Physiology
Background:
- Macrophage polarization into classically activated (M1) or alternatively activated (M2) phenotypes is crucial for regulating inflammatory responses.
- Calcium (Ca2+) signaling plays a vital role in cellular functions, including immune cell activation and differentiation.
Purpose of the Study:
- To investigate the specific roles of distinct Ca2+ entry channels in determining macrophage polarization towards M1 or M2 phenotypes.
- To elucidate the downstream signaling pathways and functional consequences regulated by these Ca2+ channels during macrophage activation.
Main Methods:
- Utilized knockout (TRPC1-/- and Orai1-/-) macrophages and mice.
- Assessed Ca2+ entry currents using electrophysiology.
- Analyzed signaling pathway activation (pNF-κB, pJNK, STAT1, STAT6) via Western blotting.
- Measured cytokine production and mitochondrial function (oxygen consumption rate, morphology).
Main Results:
- Naive and M2 macrophages rely on Orai1 for Ca2+ entry, while M1 macrophages utilize TRPC1.
- Blocking Ca2+ entry with channel inhibitors suppressed key signaling pathways and cytokine production.
- LPS stimulation induced a shift from Orai1 to TRPC1 in M2 cells, with TRPC1-/- mice showing suppressed pro-inflammatory cytokines.
- Orai1-/- macrophages exhibited reduced anti-inflammatory cytokines and impaired mitochondrial function in M2 cells.
Conclusions:
- TRPC1 and Orai1 calcium channels distinctly regulate macrophage polarization.
- These channels are critical for modulating inflammatory responses and macrophage functional phenotypes.
- Targeting TRPC1 and Orai1 offers potential therapeutic strategies for immune-related diseases.
