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Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo.

Simmons AJ, Banerjee A, McKinley ET, Scurrah CR, Herring CA, Gewin LS, Masuzaki R, Karp SJ, Franklin JL, Gerdes MJ, Irish JM, Coffey RJ, Lau KS - Mol. Syst. Biol. (2015)

Bottom Line: Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis in the seemingly homogeneous enterocyte population.Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival.Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA.

No MeSH data available.


Related in: MedlinePlus

Divergent p-ERK signaling cells are neighbors to dying cellsDistribution of p-ERK ring pattern along the whole villus, as indicated by the yellow arrows.More examples of CC3+ cells surrounded by clusters of p-ERK+ neighbors (yellow arrows).Gating of p-ERK+ cells from CyTOF data. A vehicle control (C) and a TNF-α-treated sample (C′) were used to gate for cells with homeostatic p-ERK levels versus activated p-ERK levels, respectively.p-ERK+ cells (D) and dying cells (D′) plotted in t-SNE space for cohort 1. The percentages of dying and p-ERK+ cells were used to calculate ratio of dying to p-ERK+ cells.Immunofluorescence of CC3 and p-ERK in duodenal tissue sections at 1 h post-TNF-α. CC3+ cells (green arrowheads) were flanked by p-ERK+ cells (magenta arrowheads).The efficacy of MEK inhibition assessed by p-ERK stimulation by TNF-α in the duodenum at 0.5 h. Error bars represent SEM of biological duplicates.Percentage of CC3+ cells by flow cytometry in the context of P38 inhibition. Error bars represent SEM of biological duplicates.
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fig05ev: Divergent p-ERK signaling cells are neighbors to dying cellsDistribution of p-ERK ring pattern along the whole villus, as indicated by the yellow arrows.More examples of CC3+ cells surrounded by clusters of p-ERK+ neighbors (yellow arrows).Gating of p-ERK+ cells from CyTOF data. A vehicle control (C) and a TNF-α-treated sample (C′) were used to gate for cells with homeostatic p-ERK levels versus activated p-ERK levels, respectively.p-ERK+ cells (D) and dying cells (D′) plotted in t-SNE space for cohort 1. The percentages of dying and p-ERK+ cells were used to calculate ratio of dying to p-ERK+ cells.Immunofluorescence of CC3 and p-ERK in duodenal tissue sections at 1 h post-TNF-α. CC3+ cells (green arrowheads) were flanked by p-ERK+ cells (magenta arrowheads).The efficacy of MEK inhibition assessed by p-ERK stimulation by TNF-α in the duodenum at 0.5 h. Error bars represent SEM of biological duplicates.Percentage of CC3+ cells by flow cytometry in the context of P38 inhibition. Error bars represent SEM of biological duplicates.

Mentions: Having a signaling fingerprint that classifies dying and non-dying enterocytes allows us to identify divergent signaling mechanisms that significantly affect intestinal physiology. Specifically, we chose to investigate divergent p-ERK signaling in the intestinal epithelium, which occurred in the surviving, but not in the dying, cell population. p-ERK activation in surviving enterocytes was also heterogeneous, which prompted us to envision spatial patterns of p-ERK activity that conferred survival. Whole-mount imaging of whole villus at 1 h post-TNF-α exposure revealed a “flower petal” ring-like pattern of epithelial p-ERK signaling, with five or six p-ERK-positive cells surrounding a p-ERK-negative area (Figs6A and EV5A, yellow arrows). Co-staining with CC3 revealed that in many cases, the dying CC3+ cells occupied the central area surrounded by p-ERK+ neighbors (Figs6B and EV5B, yellow arrows). In other cases, the dying CC3+ cell has already been extruded from the epithelium, leaving an apoptotic rosette surrounded by p-ERK+ cells ostensibly undergoing contraction-dependent closure (red arrow). Furthermore, the ratios of CC3+ dying cells and p-ERK+ enterocytes in three cohorts of mice were 1:4.56, 1:6.04, and 1:4.73, respectively, supporting that the immediate neighbors of the dying cell activated p-ERK signaling (FigEV5C and D). Imaging of tissue sections also corroborated that dying cells were flanked by p-ERK+ cells (FigEV5E), although the phenomenon was harder to visualize in two dimensions. We surmise that the dying cell signals to neighboring cells non-autonomously to activate a cell survival program, in order to prevent large swaths of contiguous epithelium from dying and to prevent unrecoverable barrier defects. Thus, we tested the effect of inhibiting p-ERK signaling using the allosteric MEK inhibitor PD0325901 (FigEV5F). Inhibition of p-ERK signaling affected the latency of the cell survival program such that epithelial apoptosis occurred immediately following TNF-α exposure, which resulted in a higher number of dying cells in total (Fig6C). Inhibition of P38 alone minimally affected TNF-α-induced apoptosis (Fig EV5G), but was able to partially normalize early apoptosis due to MEK inhibition (Fig6C), consistent with P38's context-dependent, pro-apoptotic role. To our knowledge, this is the first reported observation of this “flower petal” pattern of p-ERK activation in response to TNF-α-induced cell death in epithelial tissue. This new finding demonstrates the applicability of our single-cell signaling experimental platform, in conjunction with data analysis, to reveal novel, non-cell autonomous responses in complex heterogeneous epithelia.


Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo.

Simmons AJ, Banerjee A, McKinley ET, Scurrah CR, Herring CA, Gewin LS, Masuzaki R, Karp SJ, Franklin JL, Gerdes MJ, Irish JM, Coffey RJ, Lau KS - Mol. Syst. Biol. (2015)

Divergent p-ERK signaling cells are neighbors to dying cellsDistribution of p-ERK ring pattern along the whole villus, as indicated by the yellow arrows.More examples of CC3+ cells surrounded by clusters of p-ERK+ neighbors (yellow arrows).Gating of p-ERK+ cells from CyTOF data. A vehicle control (C) and a TNF-α-treated sample (C′) were used to gate for cells with homeostatic p-ERK levels versus activated p-ERK levels, respectively.p-ERK+ cells (D) and dying cells (D′) plotted in t-SNE space for cohort 1. The percentages of dying and p-ERK+ cells were used to calculate ratio of dying to p-ERK+ cells.Immunofluorescence of CC3 and p-ERK in duodenal tissue sections at 1 h post-TNF-α. CC3+ cells (green arrowheads) were flanked by p-ERK+ cells (magenta arrowheads).The efficacy of MEK inhibition assessed by p-ERK stimulation by TNF-α in the duodenum at 0.5 h. Error bars represent SEM of biological duplicates.Percentage of CC3+ cells by flow cytometry in the context of P38 inhibition. Error bars represent SEM of biological duplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig05ev: Divergent p-ERK signaling cells are neighbors to dying cellsDistribution of p-ERK ring pattern along the whole villus, as indicated by the yellow arrows.More examples of CC3+ cells surrounded by clusters of p-ERK+ neighbors (yellow arrows).Gating of p-ERK+ cells from CyTOF data. A vehicle control (C) and a TNF-α-treated sample (C′) were used to gate for cells with homeostatic p-ERK levels versus activated p-ERK levels, respectively.p-ERK+ cells (D) and dying cells (D′) plotted in t-SNE space for cohort 1. The percentages of dying and p-ERK+ cells were used to calculate ratio of dying to p-ERK+ cells.Immunofluorescence of CC3 and p-ERK in duodenal tissue sections at 1 h post-TNF-α. CC3+ cells (green arrowheads) were flanked by p-ERK+ cells (magenta arrowheads).The efficacy of MEK inhibition assessed by p-ERK stimulation by TNF-α in the duodenum at 0.5 h. Error bars represent SEM of biological duplicates.Percentage of CC3+ cells by flow cytometry in the context of P38 inhibition. Error bars represent SEM of biological duplicates.
Mentions: Having a signaling fingerprint that classifies dying and non-dying enterocytes allows us to identify divergent signaling mechanisms that significantly affect intestinal physiology. Specifically, we chose to investigate divergent p-ERK signaling in the intestinal epithelium, which occurred in the surviving, but not in the dying, cell population. p-ERK activation in surviving enterocytes was also heterogeneous, which prompted us to envision spatial patterns of p-ERK activity that conferred survival. Whole-mount imaging of whole villus at 1 h post-TNF-α exposure revealed a “flower petal” ring-like pattern of epithelial p-ERK signaling, with five or six p-ERK-positive cells surrounding a p-ERK-negative area (Figs6A and EV5A, yellow arrows). Co-staining with CC3 revealed that in many cases, the dying CC3+ cells occupied the central area surrounded by p-ERK+ neighbors (Figs6B and EV5B, yellow arrows). In other cases, the dying CC3+ cell has already been extruded from the epithelium, leaving an apoptotic rosette surrounded by p-ERK+ cells ostensibly undergoing contraction-dependent closure (red arrow). Furthermore, the ratios of CC3+ dying cells and p-ERK+ enterocytes in three cohorts of mice were 1:4.56, 1:6.04, and 1:4.73, respectively, supporting that the immediate neighbors of the dying cell activated p-ERK signaling (FigEV5C and D). Imaging of tissue sections also corroborated that dying cells were flanked by p-ERK+ cells (FigEV5E), although the phenomenon was harder to visualize in two dimensions. We surmise that the dying cell signals to neighboring cells non-autonomously to activate a cell survival program, in order to prevent large swaths of contiguous epithelium from dying and to prevent unrecoverable barrier defects. Thus, we tested the effect of inhibiting p-ERK signaling using the allosteric MEK inhibitor PD0325901 (FigEV5F). Inhibition of p-ERK signaling affected the latency of the cell survival program such that epithelial apoptosis occurred immediately following TNF-α exposure, which resulted in a higher number of dying cells in total (Fig6C). Inhibition of P38 alone minimally affected TNF-α-induced apoptosis (Fig EV5G), but was able to partially normalize early apoptosis due to MEK inhibition (Fig6C), consistent with P38's context-dependent, pro-apoptotic role. To our knowledge, this is the first reported observation of this “flower petal” pattern of p-ERK activation in response to TNF-α-induced cell death in epithelial tissue. This new finding demonstrates the applicability of our single-cell signaling experimental platform, in conjunction with data analysis, to reveal novel, non-cell autonomous responses in complex heterogeneous epithelia.

Bottom Line: Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis in the seemingly homogeneous enterocyte population.Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival.Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA.

No MeSH data available.


Related in: MedlinePlus