The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.
Quin F Wills, Esther Mellado-Gomez, Rory Nolan, Damien Warner, Eshita Sharma, John Broxholme, Benjamin Wright, Helen Lockstone, William James, Mark Lynch, Michael Gonzales, Jay West, Anne Leyrat, Sergi Padilla-Parra, Sarah Filippi, Chris Holmes, Michael D Moore, Rory Bowden
Author Information
Quin F Wills: Wellcome Trust Centre for Human Genetics (WTCHG), University of Oxford, Oxford, OX3 7BN, UK. qilin@well.ox.ac.uk.
Esther Mellado-Gomez: Wellcome Trust Centre for Human Genetics (WTCHG), University of Oxford, Oxford, OX3 7BN, UK.
Rory Nolan: Wellcome Trust Centre for Human Genetics (WTCHG), University of Oxford, Oxford, OX3 7BN, UK.
Damien Warner: Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK.
Eshita Sharma: Wellcome Trust Centre for Human Genetics (WTCHG), University of Oxford, Oxford, OX3 7BN, UK.
John Broxholme: Wellcome Trust Centre for Human Genetics (WTCHG), University of Oxford, Oxford, OX3 7BN, UK.
Benjamin Wright: Wellcome Trust Centre for Human Genetics (WTCHG), University of Oxford, Oxford, OX3 7BN, UK.
Helen Lockstone: Wellcome Trust Centre for Human Genetics (WTCHG), University of Oxford, Oxford, OX3 7BN, UK.
William James: Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK.
Mark Lynch: Fluidigm Corporation, 7000 Shoreline Ct Ste 100, South San Francisco, CA, 94080-7603, USA.
Michael Gonzales: Fluidigm Corporation, 7000 Shoreline Ct Ste 100, South San Francisco, CA, 94080-7603, USA.
Jay West: Fluidigm Corporation, 7000 Shoreline Ct Ste 100, South San Francisco, CA, 94080-7603, USA.
Anne Leyrat: Fluidigm Corporation, 7000 Shoreline Ct Ste 100, South San Francisco, CA, 94080-7603, USA.
Sergi Padilla-Parra: Wellcome Trust Centre for Human Genetics (WTCHG), University of Oxford, Oxford, OX3 7BN, UK.
Sarah Filippi: Department of Statistics, University of Oxford, Oxford, OX3 3LB, UK.
Chris Holmes: Wellcome Trust Centre for Human Genetics (WTCHG), University of Oxford, Oxford, OX3 7BN, UK.
Michael D Moore: Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK. kenny.moore@path.ox.ac.uk.
Rory Bowden: Wellcome Trust Centre for Human Genetics (WTCHG), University of Oxford, Oxford, OX3 7BN, UK. rbowden@well.ox.ac.uk.
BACKGROUND: Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS: We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. CONCLUSION: As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.
