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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session A

Poster G1 - Autofluorescence Lifetime Imaging of Metabolism in Progenitor-derived Neutrophils

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Aleksandr Kralovec

Electrical Engineering Student
University of Wisconsin - Madison
De Pere, Wisconsin, United States

Co-Author(s)

RD

Rupsa Datta, PhD

Associate Scientist
Morgridge Institute for Research, United States
JV

James Votava

Postdoctoral Fellow
Morgridge Institute for Research, United States
JF

Jing Fan

Investigator
Morgridge Institute for Research, United States

Primary Investigator(s)

MS

Melissa Skala, PhD

Professor
University of Wisconsin - Madison, United States

Introduction: Neutrophils are the most abundant white blood cells in the body; they play a critical role in defending against pathogens, foreign bodies, and cancer. In contrast to other immune cells, neutrophils possess a very short lifespan in culture; this renders many experimental tools ineffective. Thus, primary (blood-derived) neutrophils are often substituted with a cell-line model that can be cultured over extended periods of time. For instance, the HL-60 promyelocytic cell line is used to investigate the functional, morphological, and metabolic characteristics of primary neutrophils. While studies have validated the functional and morphological similarities between primary neutrophils and differentiated HL-60s (dHL-60s), little research has been conducted comparing the metabolic properties of primary neutrophils to dHL-60s, specifically at the single-cell level. In this work we perform single-cell metabolic characterization of dHL-60s using fluorescence lifetime imaging microscopy (FLIM), a non-invasive, dye/label free, multiphoton microscopy technique that quantifies the fluorescent lifetimes (τ) of nicotinamide adenine dinucleotide phosphate (NAD(P)H). These lifetimes depend highly upon its binding state, conformations, and surrounding microenvironment. For example, free NAD(P)H has a shorter lifetime than enzyme-bound NAD(P)H. Therefore, NAD(P)H FLIM can be used to characterize the metabolic states of cells [1].
In addition to FLIM, neutrophil functions, such as NETosis, were measured in dHL-60s to validate their activation. Quantifying the metabolic shifts during activation will help better understand and characterize the progenitor cell derived neutrophil cell line, consequently providing opportunities to improve their function and facilitate further therapeutic development.

Materials and

Methods: Cell Culture:
HL-60s were cultured in RPMI media with 25 mM HEPES, 5 mM L-Glutamine, and 15% FBS at 37°C and 5% CO2. Following differentiation in 1.3% dimethyl sulfoxide (120 hours), dHL-60s were washed and plated in dishes (400,000/50 uL) coated with P-selectin. Neutrophils from healthy human donors were isolated via negative selection with the MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec). Cells were activated with either 100 nM phorbol myristate acetate (PMA) or 4 µM Ionomycin for 15 & 20 minutes, respectively, and inhibited with 100 nM diphenylene iodonium (DPI).
Imaging:
FLIM was performed using a custom multi-photon microscope (Bruker). NAD(P)H was excited at 750 nm and emission was collected by GaAsP photomultiplier tubes (Hamamatsu) through a bandpass filter 440/80 nm. Lifetime measurements were acquired using time-correlated single-photon counting electronics (Becker & Hickl); decays were fitted to a biexponential model [I(t) = α1 exp(−τ/ τ1) + α2 exp(−τ/τ2) + C)]. I(t): fluorescence intensity at time t; α1, α2: fractional contributions; τ1, τ2: the short and long lifetime components, respectively; C: background light, using SPCImage (Becker & Hickl GmbH). Mean lifetime, τm = α1 τ1 + α1 τ2. Single-cell segmentation was performed via CellPose [2]. dHL-60s without multi-lobed nuclei were manually excluded. Results were examined for statistical significance with GraphPad.
NETosis:
dHL-60s were incubated with Incucyte® Cytotox Dye (Sartorius), a green, fluorescent dye, plated at 50,000 cells/uL, and imaged over 36 hours (1–2-hour intervals), via the Incucyte fluorescence imager. Average green area per well was plotted in GraphPad.

Results, Conclusions, and Discussions:

Results:
To investigate the metabolic properties of dHL-60s compared to primary neutrophils we performed NAD(P)H FLIM. Upon treatment with PMA, a chemical protein kinase C stimulus, both dHL-60s and primary neutrophils show a significant decrease in NAD(P)H τm and increase in NAD(P)H α1 (percentage of free NAD(P)H) (a, b, c). This corresponds to the known activation of metabolic pathways, specifically glycolysis and Pentose Phosphate Pathway (PPP). Moreover, with NAD(P)H oxidase (NOX) inhibitor DPI, we observed that primary neutrophils and dHL-60s exhibit symmetrical behavior with respect to NAD(P)H τm, and NAD(P)H α1 (a,b,c).
To further assess the metabolic pathways critical for dHL-60s, cells were treated with two functionally different activators, PMA and Ionomycin [3-4]. As shown in (d), dHL-60s stimulated with PMA present a large decrease in NAD(P)H τm compared to unstimulated controls. On the contrary, ionomycin did not elicit notable changes. Additionally, as seen in (e) dHL-60s activated with PMA released a significant quantity of neutrophil extracellular traps (NETs) while both control and Ionomycin-stimulated cells did not. Likewise, co-treatment with PMA and DPI caused lower NET production.

Discussion:
Our results indicate similar metabolic responses to PMA in dHL-60s and primary neutrophils, with a transition from oxidative metabolic pathways (i.e., electron transport chain) to glycolytic pathways (i.e., glycolysis, PPP). Additionally, NAD(P)H τm, supported by NETosis assay results, suggest that dHL-60s may have a larger dependence on the PPP for activation and NOX2 generated reactive oxygen species (ROS) production as compared to other mitochondrial pathways, such as those activated by ionomycin. However, further experiments with additional metabolic activators and inhibitors are required to gain a more complete understanding of the activation requirements of dHL-60s.

Conclusion:
We characterized dHL-60s as a model for neutrophil metabolism using NAD(P)H FLIM and functional measurements of NETosis. Overall, this study provides insights into metabolic requirements during activation, along with the functionality of progenitor-derived neutrophils. A better understanding of these metabolic and functional features may help identify molecular targets for treatments of neutrophil related diseases and blood cancers.

Acknowledgements (Optional):

References:
[1] Rupsa Datta, et al., "Recent innovations in fluorescence lifetime imaging microscopy for biology and medicine," J. Biomed. Opt. 26(7) 070603 (10 July 2021)
[2] C. Stringer, T. Wang, et all., “Cellpose: a generalist algorithm for cellular segmentation,” Nature Methods, vol. 18, no. 1, pp. 100–106, Jan. 2021, doi: https://doi.org/10.1038/s41592-020-01018-x.
[3] G. Morán, B. Uberti, and J. Quiroga, “Role of Cellular Metabolism in the Formation of Neutrophil Extracellular Traps in Airway Diseases,” Front. Immunol., vol. 13, Apr. 2022, doi: 10.3389/fimmu.2022.850416.
[4] H. L. Damascena, W. A. A. Silveira, M. S. Castro, and W. Fontes, “Neutrophil Activated by the Famous and Potent PMA (Phorbol Myristate Acetate),” Cells, vol. 11, no. 18, p. 2889, Sep. 2022, doi: https://doi.org/10.3390/cells11182889.