Towards modelling metabolic state from single-cell transcriptomics

Abstract DNA damage and the attendant cellular responses of apoptosis, senescence, and altered differentiation are major drivers of hematopoietic stem cell (HSC) aging. A reservoir of persistent DNA damage signaling can derive from progressive telomere erosion, which occurs over the lifespan of humans. However, the molecular mechanisms by which telomere damage compromises HSC functions are largely unknown. Here, though combined single-cell RNA-seq and functional studies of highly-purified c-Kit+Sca+Lin-CD34-flk2-CD150+CD48-CD41-HSCs, we show that persistent telomeric damage does not activate programs of apoptosis or senescence but maintains HSCs in an activated metabolic state, which directly compromises their self-renewal capability. To dissect the biological and molecular mechanisms by which persistent DNA damage affects HSC function we analyzed the HSC compartment of mice with short telomeres (G5/G6 TERTER/ER), which developed age-related defects. Immunophenotypic analysis of the HSC compartment showed that, compared with G0 TERTER/+ (G0) mice with intact telomeres (n=12), 2 month-old G5/G6 mice (n=17), had a significantly decreased number of HSCs (p<10-3) that was associated with a decreased number of the lymphoid-biased MPP4 cells, and an increased number of both megakaryocyte-biased MPP2 and myeloid-biased MPP3 cells. HSC exhaustion and increased myeloid-to lymphoid output were reminiscent of stressed hematopoiesis and premature aging. G5/G6 HSCs exhibited a significant accumulation of telomere dysfunction-induced foci (p<10-5) but did not display increased levels of apoptosis in steady-state conditions. HSC exhaustion could result from apoptosis and/or senescence induced by telomere damage in HSCs entering the cell cycle or from an altered balance between self-renewal and differentiation. To distinguish between these two possibilities, we first investigated the effect of inducing young G5/G6 HSCs out of a homeostatic quiescent state. By tracking the real-time changes in the expression level of annexin V on HSCs induced to differentiate towards the myeloid lineage, we found that apoptosis was not the primary fate of G5/G6 HSCs upon entry into the cell cycle. Similarly, in vivo treatment with poly I:C induced the G5/G6 HSCs to enter into the cell cycle at the same rate as that of the G0 mice without inducing apoptosis. Transcriptomic analysis of poly I:C-treated G0 and G5/G6 HSCs, compared with vehicle-treated controls, revealed a significant enrichment of genes involved in the regulation of the cell cycle and platelet production, which is consistent with previous findings showing that megakaryocyte differentiation of HSCs occurs in response to poly I:C to replenish platelets that are lost during inflammatory insult. Importantly, we did not observe any significant change in gene expression between G0 and G5/G6 HSCs isolated from poly I:C-treated mice, which confirmed that telomeric damage did not limit HSCs' proliferation potential by activating programs of senescence or apoptosis. Next, we evaluated the capability of single HSCs isolated from G0 or G5/G6 mice to either self-renew or differentiate. An evaluation of Numb inheritance and expression in G0 and G5/G6 HSCs (n=133 and n=113, respectively) induced to proliferate in vitro showed that G5/G6 HSCs had a 2-fold lower frequency of symmetric self-renewal division (p<10-3) and a concomitant 2-fold higher frequency of symmetric commitment (p<10-4). Accordingly, PB analysis revealed that the CD45.2-derived reconstitution was severely compromised in mice competitively transplanted with G5/G6 HSCs (0.26% vs 77%; p<10-3). Single cell RNA-seq analysis of G0 and G5/G6 HSCs followed by the differential analysis of the clusters showed that 40% of G5/G6 HSCs were in an activated metabolic state associated with hyperactive OXPHOS and ROS signaling pathways, which are directly involved in HSC functional decline. Finally, we reactivated telomerase to investigate the possibility of restoring normal HSC function upon elimination of damage. Single cell RNA-seq and functional studies are ongoing to evaluate whether HSCs' activated metabolic state and compromised self-renewal capability are reversible processes. This study challenges the concept that telomeric damage limits HSC's proliferative potential and offers unparalleled opportunities for unraveling regenerative strategies to ameliorate their decline. Disclosures Colla: Abbvie: Research Funding.

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Single-cell metabolic dynamics of early activated CD8 T cells during the primary immune response to infection

10.1101/2020.01.21.911545 ◽

2020 ◽

Cited By ~ 1

Author(s):

Lauren S. Levine ◽

Kamir J. Hiam ◽

Diana M. Marquez ◽

Iliana Tenvooren ◽

Diana C. Contreras ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Single Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Primary Immune Response ◽

Flux Analysis ◽

Metabolic State ◽

Activated T Cells

AbstractMemory T cells conventionally rely on oxidative phosphorylation and short-lived effector T cells on glycolysis. Here, we investigate how T cells arrive at these states during an immune response. In order to understand the metabolic state of rare, early activated T cells, we adapted mass cytometry to quantify metabolic regulators at single-cell resolution in parallel with cell signaling, proliferation, and effector function. We interrogated CD8 T cell activation in vitro as well as the trajectory of CD8 T cells responding to Listeria monocytogenes infection, a well-characterized in vivo model for studies of T cell differentiation. This approach revealed a unique metabolic state in early activated T cells characterized by maximal expression of glycolytic and oxidative metabolic proteins. Peak utilization of both pathways was confirmed by extracellular flux analysis. Cells in this transient state were most abundant five days post-infection before rapidly downregulating metabolic protein expression. This approach should be useful for mechanistic investigations of metabolic regulation of immune responses.

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A novel strategy for single-cell metabolic analysis highlights dynamic changes in immune subpopulations

10.1101/2020.01.21.914663 ◽

2020 ◽

Cited By ~ 1

Author(s):

Patricia J. Ahl ◽

Richard A. Hopkins ◽

Wen Wei Xiang ◽

Bijin Au ◽

Nivashini Kaliaperumal ◽

...

Keyword(s):

Single Cell ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Cell Subset ◽

Metabolic Reprogramming ◽

Metabolic State ◽

Peripheral Blood Mononuclear ◽

Metabolic Remodeling ◽

Novel Strategy ◽

Metabolic Heterogeneity

SummaryA complex interaction of anabolic and catabolic metabolism underpins the ability of leukocytes to mount an immune response. Their capacity to respond and adapt to changing environments by metabolic reprogramming is crucial to their effector function. However, current methods lack the ability to interrogate this network of metabolic pathways at the single cell level within a heterogeneous population. Here we present Met-Flow, a novel flow cytometry-based method that captures the metabolic state of immune cells by targeting key proteins and rate-limiting enzymes across multiple pathways. We demonstrate the ability to simultaneously measure divergent metabolic profiles and dynamic remodeling in human peripheral blood mononuclear cells. Using Met-Flow, we discovered that glucose restriction and metabolic remodeling drive the expansion of an inflammatory central memory T cell subset. This method captures the complex metabolic state of any cell as it relates to its phenotype and function, leading to a greater understanding of the role of metabolic heterogeneity in immune responses.

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Uncovering metabolic states in cytotoxic T cells, one cell at a time

Science Translational Medicine ◽

10.1126/scitranslmed.abe6027 ◽

2020 ◽

Vol 12 (563) ◽

pp. eabe6027

Author(s):

Ioannis Zervantonakis

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Cytotoxic T Cells ◽

Activity Patterns ◽

Cell Activity ◽

Cytotoxic T Cell ◽

Colorectal Tumors ◽

Metabolic State ◽

Metabolic States

Single-cell metabolic state analysis reveals cytotoxic T cell activity patterns that are spatially organized in human colorectal tumors.

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Electron microscopy of keratinocytes cultured on a collagen substrate used as an allograft for the treatment of chronic wounds

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100130158 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1104-1105

Author(s):

Debby A. Jennings ◽

Michael J. Morykwas ◽

Louis C. Argenta

Keyword(s):

Electron Microscopy ◽

Cell Suspension ◽

Single Cell ◽

Chronic Wounds ◽

Mechanical Stability ◽

Uv Light ◽

Type I ◽

Single Cell Suspension ◽

Collagen Substrate ◽

Dermal Collagen

Grafts of cultured allogenic or autogenic keratlnocytes have proven to be an effective treatment of chronic wounds and burns. This study utilized a collagen substrate for keratinocyte and fibroblast attachment. The substrate provided mechanical stability and augmented graft manipulation onto the wound bed. Graft integrity was confirmed by light and transmission electron microscopy.Bovine Type I dermal collagen sheets (100 μm thick) were crosslinked with 254 nm UV light (13.5 Joules/cm2) to improve mechanical properties and reduce degradation. A single cell suspension of third passage neonatal foreskin fibroblasts were plated onto the collagen. Five days later, a single cell suspension of first passage neonatal foreskin keratinocytes were plated on the opposite side of the collagen. The grafts were cultured for one month.The grafts were fixed in phosphate buffered 4% formaldehyde/1% glutaraldehyde for 24 hours. Graft pieces were then washed in 0.13 M phosphate buffer, post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Polybed 812.

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