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Spatial confinement downsizes the inflammatory response of macrophages

Nature Materialsvolume 17pages11341144 (2018) | Download Citation

Abstract

Macrophages respond to chemical/metabolic and physical stimuli, but their effects cannot be readily decoupled in vivo during pro-inflammatory activation. Here, we show that preventing macrophage spreading by spatial confinement, as imposed by micropatterning, microporous substrates or cell crowding, suppresses late lipopolysaccharide (LPS)-activated transcriptional programs (biomarkers IL-6, CXCL9, IL-1β, and iNOS) by mechanomodulating chromatin compaction and epigenetic alterations (HDAC3 levels and H3K36-dimethylation). Mechanistically, confinement reduces actin polymerization, thereby lowers the LPS-stimulated nuclear translocation of MRTF-A. This lowers the activity of the MRTF-A–SRF complex and subsequently downregulates the inflammatory response, as confirmed by chromatin immunoprecipitation coupled with quantitative PCR and RNA sequencing analysis. Confinement thus downregulates pro-inflammatory cytokine secretion and, well before any activation processes, the phagocytic potential of macrophages. Contrarily, early events, including activation of the LPS receptor TLR4, and downstream NF-κB and IRF3 signalling and hence the expression of early LPS-responsive genes were marginally affected by confinement. These findings have broad implications in the context of mechanobiology, inflammation and immunology, as well as in tissue engineering and regenerative medicine.

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Data availability

Microarray data for HDAC3-KO BMDMs were obtained from ref. 32 and the GEO accession no. is GSE33162. RNA–seq data for Control untreated BMDMs, LPS-treated BMDMs and SRF-KO-LPS-treated BMDMs have been deposited in GEO. The accession codes are GSM3373977–GSM3373979, GSM3373980–GSM3373982 and GSM3373983–GSM3373985 for Control untreated BMDMs, LPS-treated BMDMs and SRF-KO-LPS-treated BMDMs, respectively. The remaining data supporting the findings of this study are available within the Article and its Supplementary Information files and from the corresponding authors upon reasonable request.

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Acknowledgements

The authors thank A. Oxenius and the Mice Facility both at ETH Zurich for donating postmortem animals for bone marrow isolation, S. Farmer (Boston University) for providing bone marrow from MRTF-A-KO mice and for helpful discussions, S. Halene (Animal Modeling Core, Yale Cooperative Center of Excellence in Hematology (YCCEH) NIDDK grant no. U54DK106857, Yale University) for providing bone marrow from SRF-KO mice, and K. Sobue and T. Mayanagi (Iwaka Medical University) for providing CA-MRTF-A plasmid. The authors thank G. V. Shivashankar (MBI, NUS, Singapore) for providing HDAC3-overexpressing plasmid, L. Philipp and U. Kutay (ETH Zurich) and M. O. Hottiger (University of Zurich) for helpful discussions on ChIP experiments. In the Vogel group, I. A. Vizcarra is acknowledged for initial help with BMDM culture, J.-Y. Shiu and V. Hosseini for their advice and the manufacturing of 3D-microwells, C. Spencer for technical support, I. Gerber for acquiring the electron microscopy images, L. A. Branco and I. Schoen for critical comments on the manuscript and finally the FIRST and ScopeM facilities at ETH Zurich for access to microfabrication and confocal microscopy. This work was supported by ETH Zurich and by the Swiss National Science Foundation Grant (grant no. CR32I3_156931 to V.V.) and also in part by the Swiss NCCR Molecular Systems Engineering.

Author information

Affiliations

  1. Laboratory of Applied Mechanobiology, Institute of Translational Medicine, Department of Health Sciences and Technology, ETH Zurich, Switzerland

    • Nikhil Jain
    •  & Viola Vogel

Authors

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Contributions

N.J. and V.V. conceived of the project. N.J designed and performed the experiments. N.J. and V.V. analysed the data and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Nikhil Jain or Viola Vogel.

Supplementary information

  1. Supplementary Information

    Supplementary Video Legends 1–5, Supplementary Figures 1–22, Supplementary Tables 1,2, Supplementary Reference 1

  2. Reporting Summary

  3. Supplementary Data 1

    HDAC3-KO microarray analysis

  4. Supplementary Data 2

    Gene ontology analysis of control LPS-treated BMDMs versus control untreated BMDMs

  5. Supplementary Data 3

    RNA-Seq analysis and gene ontology analysis of SRF-KO LPS-treated BMDMs versus control LPS-treated BMDMs

  6. Supplementary Video 1

    Spreading of control BMDMs

  7. Supplementary Video 2

    Spreading of LPS-treated BMDMs

  8. Supplementary Video 3

    Spreading of IL-4/IL-13-treated BMDMs

  9. Supplementary Video 4

    3D reconstruction of unconfined (UC) BMDM treated with LPS for 6 h and stained for nuclei, F-actin, G-actin and MRTF-A

  10. Supplementary Video 5

    3D reconstruction of confined (CC) BMDMs treated with LPS for 6 h and stained for nuclei, F-actin, G-actin and MRTF-A

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DOI

https://doi.org/10.1038/s41563-018-0190-6

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