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Dissonant response of M0/M2 and M1 bone-marrow-derived macrophages to RhoA pathway interference

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Abstract

Macrophages have a multitude of functions in innate and adaptive immune response and organ and tissue homeostasis. Many experimental studies are performed on bone-marrow-derived macrophages differentiated in vitro into M1 (inflammatory) and M2 (anti-inflammatory) subtypes that express different molecular markers pertaining to their prospective functions. Macrophage phenotype, polarity and functions depend on the actin cytoskeleton, which is regulated by small GTPase RhoA, its downstream effector ROCK, and non-apoptotic Caspase-3. We generated transgenic mice with the macrophage-specific deletion of RhoA and compared the effect of Rho pathway interference (RhoA deletion and ROCK and Caspase-3 inhibition) on the phenotype, polarity and expression of subtype-specific molecular markers of bone-marrow-derived M0, M1 and M2 macrophages. We show that M0 and M2 macrophages have a radically different phenotype and polarity from M1 macrophages, and that this is mirrored in dissonant response to RhoA pathway interference. The RhoA pathway interference induces extreme elongation (hummingbird phenotype) of M0 and M2 but not M1 macrophages and inhibits the expression of M2-specific but not M1-specific molecular markers. These dramatic differences in the response of M0/M2 versus M1 macrophages to the same molecular cues ought to be important considerations in the interpretation of experimental data and therapeutic use of bone-marrow-derived macrophages.

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Abbreviations

Arg 1:

Arginase

CCL2:

Chemokine (C-C motif) ligand 2

FA:

Focal adhesion

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

iNOS:

Inducible nitric oxide synthase

Q-RTPCR:

Quantitative real-time polymerase chain reaction

RhoA:

Ras homolog gene family, member A

ROCK:

Rho-associated protein kinase (ROCK1 and ROCK2)

TNFα:

Tumor necrosis factor, a cytokine that can induce cell death (apoptosis)

Y-27632:

Selective ROCK1 (p160ROCK) inhibitor

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Authors and Affiliations

  1. The Houston Methodist Research Institute, Houston, Tex., USA

    Yianzhu Liu, Wenhao Chen, Laurie J. Minze, Xian C. Li, Rafik M. Ghobrial & Malgorzata Kloc

  2. Department of Surgery, The Houston Methodist Hospital, 6550 Fannin Street, Houston, TX 77030, USA

    Yianzhu Liu, Wenhao Chen, Laurie J. Minze, Xian C. Li, Rafik M. Ghobrial & Malgorzata Kloc

  3. Department of Neurology, Xiangya Hospital, Central South University, Changsha, 410008 Hunan, China

    Yianzhu Liu

  4. CNRS UMR 6290, Institute of Genetics and Development of Rennes, Cell Cycle Group, IFR 140 GFAS, Rennes, France

    Jacek Z. Kubiak

  5. University of Rennes 1, Faculty of Medicine, 35043, Rennes, France

    Jacek Z. Kubiak

  6. Sherrie and Alan Conover Center for Liver Disease and Transplantation, Houston, Tex., USA

    Rafik M. Ghobrial

Authors
  1. Yianzhu Liu
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  2. Wenhao Chen
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  3. Laurie J. Minze
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  4. Jacek Z. Kubiak
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  5. Xian C. Li
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  6. Rafik M. Ghobrial
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  7. Malgorzata Kloc
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Corresponding author

Correspondence to Malgorzata Kloc.

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The authors of this manuscript have no conflicts of interest to disclose.

Additional information

We are grateful for the support from the William Stamps Farish Fund and the Donald D. Hammill Foundation.

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ESM 1

Quantitative analysis of macrophage length in M0 and M2 subtypes. a M0 and M2 macrophages have a similar percentage of elongated macrophages. b M0 RhoA-deleted (KO) and M2 RhoA-deleted (KO) macrophages have a similar percentage of elongated macrophages. c M0 and M2 macrophages treated with Y-27632 inhibitor have a similar percentage of elongated macrophages. d M0 and M2 macrophages subtypes treated with Y-27632 inhibitor have a similar percentage of elongated (average length: 164 μm) and extremely elongated (average length: 364 μm) macrophages with P = 0.7673 and 0.7106, respectively. However, within each subtype, the percentage of extremely elongated macrophages was significantly higher than elongated macrophages. P-value = 0.0077 and 0.0015, respectively. Statistically significant differences are P < 0.05. The calculations were performed on 100 macrophages in 10 different quadrants of microscope slides. (PDF 660 kb)

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Liu, Y., Chen, W., Minze, L.J. et al. Dissonant response of M0/M2 and M1 bone-marrow-derived macrophages to RhoA pathway interference. Cell Tissue Res 366, 707–720 (2016). https://doi.org/10.1007/s00441-016-2491-x

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  • DOI: https://doi.org/10.1007/s00441-016-2491-x

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