- Assess macrophage M1 activation in response to pro-inflammatory mediators
- Livecyte's high-contrast label-free imaging and robust single-cell segmentation enable automatic quantification of cell morphology
- Growth and proliferation changes over time show the time-dependent nature of pro-inflammatory macrophage activation
The inflammatory process is stringently controlled and involves numerous chemical signals to initiate, maintain and resolve inflammation. Macrophages are an essential part of this system and regulate both the innate and adaptive immune system . Once activated, resident macrophages act as a first line of defence against pathogens and are involved in phagocytosis and clearance of microbes and apoptotic cells. Furthermore, they play a fundamental role in initiating and regulating the host adaptive immune response by communicating with B and T lymphocytes through the release of cytokines and cell-cell interactions .
Multiple soluble factors in the tissue microenvironment can influence macrophage activation or ‘polarisation’ towards specific functional initiatives. In the presence of inflammatory signals such as Interferon γ (IFNγ) and bacterial lipolysaccaride (LPS), macrophages move towards a classical M1 phenotype . Once activated to an M1 phenotype, macrophages produce a variety of biologically active mediators which engage in both favourable and detrimental outcomes to inflammation. M1 macrophages gain microbicidal properties and release pro-inflammatory cytokines such as IL-1ᵦ and TNF-α as well as chemokines to recruit other immune cells, particularly T lymphocytes to the site of infection.
Within the immune system, a complex interplay exists between macrophages and T lymphocytes. The presence of an inflammatory stimuli, such as bacterial LPS on the surface of microbes, are detected by receptors on the surface of nearby macrophages and trigger a morphologic M1 phenotype . Once identified, macrophages phagocytose bacteria and present microbial peptides on their cell surface. These are recognised by differentiated effector T lymphocytes which in turn become activated. Activated T lymphocytes release IFNγ which leads to further activation of adjacent macrophages and advance the macrophage activation cascade .
Through these actions macrophages can coordinate the development of chronic inflammatory diseases including the progression of pathophysiological conditions such as cancer, cardiovascular diseases  and type II diabetes . Consequently, activated macrophages and their products may be useful therapeutic targets to influence outcomes in inflammatory diseases.
Livecyte utilises Ptychography, a quantitative phase imaging (QPI) technique, to produce high-contrast images without the need for fluorescent labels. The enhanced contrast enables automatic segmentation and tracking of individual cells, as well as a quantitative measure of proliferation and morphology of cells over time.
In this study the macrophage cell line RAW 264.7 was successfully activated using the proinflammatory mediators LPS and IFNγ. Proliferation and cell and population morphology were investigated through Livecyte's label-free QPI mode and in-built Analyse Software.
RAW 264.7 macrophages were routinely maintained in DMEM supplemented with 10% FBS (complete medium hereafter) at 37°C with 5% CO2/95% humidity prior to experiments. Cells were harvested using standard techniques and cell count and viability was determined by trypan blue exclusion (ViCell; Beckman Coulter®). Cells were seeded into the wells of a 96 well plate at 10,000 cells/well and cultured overnight. Media was replaced with complete medium only or complete medium containing LPS (10ng/ml) and/or IFNγ (10ng/ml) and incubated 1 hour prior to imaging.
• RAW 264.7 macrophage cell line
• DMEM (Gibco)
• Foetal Bovine Serum (FBS; Gibco)
• LPS and IFNγ (10ng/mL)
• 96-well culture plate (Corning® 3603)
• Livecyte Kinetic Cytometer (Phasefocus)
• Livecyte Acquire & Analyse software (Phasefocus)
Changes in morphology were assessed with the Livecyte™ system's unique label-free Quantitative Phase Imaging (QPI). Cells were imaged with an Olympus PLN 10X (0.25NA) objective and 1mm x 1mm field of view (FOV) per well for 40 hours at 15 minute intervals. Cells were maintained inside an environmental chamber at 37°C with 5% CO2/95% humidity.
Analysis & Results
Quantitative Phase Images & Cell Segmentation
The quantitative phase images generated by Livecyte are high contrast and illustrative images from the different conditions are shown below (figure 1).
High contrast images enable robust cell segmentation and analysis of subtle changes in cell shape, size, and mass as well as tracking over long time periods. For instance, these label-free images show cells exposed to LPS became larger and flatter with small filopodial projections around the edges of the cell membrane compared to control cells (figure 1c). These findings conform with the literature that suggest macrophages in the proinflammatory phenotype have an increased cell area to accommodate binding and phagocytosis of foreign bodies  and gain multiple extensions to allow cells to adhere at the site of infection. Furthermore, high contrast images allowed us to observe subcellular changes particularly an increase in vacuole formation .
Phagocytic vacuoles traditionally enclose foreign particles which can then be digested with hydrolytic enzymes during an immune response suggesting LPS increases macrophage microbicidal activity . These morphological changes were exacerbated when cells were treated with both LPS and IFNγ (figure 1d) but not with treatment of IFNγ alone (figure 1b). Previous studies have shown that although IFNγ and LPS co-treatment enhanced macrophage pro-inflammatory activity i.e. an increase in cytokine secretion, treatment with IFNγ without LPS did not produce this response . The underlying mechanisms of this priming effect are unclear; however, it is presumed IFNγ co-treatment may lead to transcriptional/posttranscriptional changes within macrophages which upregulate LPS receptors on cells subsequently leading to a more robust inflammatory response as well as enhanced expression of downstream response factor genes .
From the time-lapse images we discerned a change in morphology whereby, in response to IFNγ and LPS, cells became larger and flatter and produced multiple projections. To gain a further insight in and quantify these phenotypic changes, we used metrics derived from the Morphology Dashboard in Livecyte’s Analyse software. Cells incubated with LPS and a combination of both LPS and IFNγ showed a considerable increase in area and perimeter (figure 2a&b). This was particularly striking in the LPS/IFNγ treatment group and suggested macrophages were activated to a greater extent in this group. Further scrutiny of these treatment groups revealed that at population level this response to LPS and IFN was time sensitive. An exponential increase in both area and perimeter was noted after ~6 hours after which a plateau was reached at ~18 hours. This suggests macrophages are most active in the first 24 hours post-treatment and that either the amount of
pro inflammatory mediator may be the rate limiting factor or indeed other regulatory pathways may be controlling the macrophage immune response. One known regulatory pathway is a suppressor loop involving IL-10 production by macrophages after activation with LPS. This inhibits the long-term production of inflammatory cytokines and may prevent the over-activation and prolonged M1 activation of macrophages . In addition to an increase in area and perimeter a concurrent decrease in sphericity was also observed at both single cell and population level. This followed a similar trend to that of cell area and perimeter signifying that macrophages were becoming larger, and lengthening, producing multiple projections (figure 2c).
Cell Proliferation and Growth
Investigating proliferation and growth of RAW 264.7 cells showed subtle time-sensitive changes in cell count and proliferation in cells treated with LPS and/or IFNγ. At ~6 hours a plateau in cell count was observed combined with an overall increase in cell doubling time (figure 3a&b). It was also possible to study growth of cells through calculating total dry mass. Dry mass is defined as the cellular biomatter within cells minus water and therefore growth of cells can be identified by an accumulation of biomass. A slight reduction in the rate of total dry mass was seen in cells treated with LPS and/or IFNγ however this was not proportional to the cell count (figure 3c). In contrast to the cell count plot, no plateau was observed at the beginning of the time lapse in the dry mass data. This may be a possible indication of a prioritisation of energy and resources towards a rapid inflammatory response suppressing proliferation pathways and focusing on producing proinflammatory mediators .
Single cell level dry mass analysis (figure 4) showed RAW 264.7 cells treated with either LPS alone or in combination with IFNγ increased in dry median mass after 6 hours suggesting a suppression of the proliferation pathway led to an accumulation of dry mass at the single cell level. A plateau in dry mass was reached at around 18 hours at which point the rate of proliferation increased possibly
due to mediators being used up or a negative feedback loop reducing escalation of the inflammatory response. A strong correlation between these temporal growth and proliferation changes and morphological changes seen in cells at this time suggested a window of RAW 264.7 cell activation between 6-18 hours.
Figure 1: Quantitative phase images of a) untreated macrophages and those treated with b) IFNγ (10ng/ml), c) LPS (10ng/ml) and d) a combination of both. Images were taken at x20 magnification at 40hrs and illustrate the cellular and sub-cellular morphological changes observed.
Figure 2: Graphs taken from the Livecyte Analyse Morphology Dashboard illustrating changes in Median Cell Area over time (a) Median Cell Perimeter (b) and Median Cell Sphericity (c)for each of the treatment groups and control. Both Area and Perimeter show temporal changes with an increase at ~8 hours after which a plateau was reached at around 24 hours. Sphericity of the cells was shown to decrease during this time after which cells also started to plateau.
Figure 3: Illustrative graphs from the Proliferation Dashboard. a) Cell Count plot shows a plateau at 6 hours with an overall reduction in CellDoubling Time (b) in RAW264.7 cells treated with either LPS alone or LPS in combination with IFNγ. However Total Dry Mass analysis showed a constant dry mass accumulation during this time (c). An overall increase in Dry Mass Doubling Time was observed in these treated cells however this not proportional to the Cell Doubling Time (d).
Figure 4: Illustrative graph from the Morphology Dashboard. Median Cell Dry Mass plot showed an increase in dry mass at ~6 hours in RAW cells treated with LPS and/or IFNγ.
Macrophages play a key role in the initiation and regulation of inflammation during infection but also in chronic inflammatory diseases. They’re coordinated response working as part of the innate and adaptive immune system mean they interact and respond to both foreign antigens and adaptive immune cell mediators. This leads to changes in their phenotype making them better equipped to perform their role .
Quantitative phase imaging generates high-contrast images that enable robust cell segmentation and quantification of morphology metrics. Coupled with time-lapse imaging and tracking algorithms the Livecyte provides users with the tools to monitor how cells alter their morphology over time, independently in response to external conditions.
In this study we characterised subtle changes in macrophage phenotype in response to inflammatory stimuli. Through analysing cell count and cell dry mass values we were able to identify proliferation and growth of cells independently. We identified a prioritisation towards proinflammatory signalling and growth with an inhibition of the proliferation pathway in cells treated with LPS. This is known to occur in activated macrophages to meet the demands of cell growth and production of bactericidal factors . This was exacerbated with the addition of pro-inflammatory cytokine IFNγ suggesting a synergistic effect of both these pro-inflammatory mediators.
We also report that incubation of cells with LPS and/or IFNγ showed all the hallmark phenotypic changes of classical M1 activation with both an increase in cell area and perimeter. In addition, through quantitative phase imaging and accurate cell segmentation we were able to identify an increase in small filopodial projections and quantify this through a reduction in cell sphericity. No change was seen in cells treated with IFNγ alone, suggesting M1 activation is largely LPS dependant.
Importantly both these changes in morphology, cell proliferation and growth were time-sensitive and coincided with each other. A maintenance in cell growth and reduction the rate of proliferation was accompanied with an increase in area and perimeter at around 6 hours. After a period of 16-24 hours a plateau in these metrics was observed. Previously, real-time investigation of RAW 264.7 cell activation with LPS/IFNγ through cellular impedance showed a similar response pattern whereby around a 10 hour lag in response was observed after which a peak in impedance was observed at around 18 hours. Impedance values declined after 24 hours suggesting an optimum period of pro-inflammatory activation was seen between 6- 24 hours in these cells
. Although this study highlights and corroborates the temporal regulation of macrophage function we found, this study was solely investigating changes in morphology through cellular impedance and therefore was unable to distinguish and quantify exact feature changes without further confocal measurements. In vitro immune assays although extensively used to mimic infection conditions can suffer from lack of physiological relevance, partly through the lack of temporal analysis and a reliance on limited metrics leading to insufficient immune cell characterisation. Through Livecyte’s quantitative phase imaging mode and Analyse software we were able to automatically extract a wealth of single cell and population metrics label free. In
being able to investigate and measure several characteristics of cells, Livecyte provides a promising tool for investigating innate immune response regulation. In combination with an intuitive workflow, Livecyte enabled us to reliably investigate the time-sensitive phenotypic changes of activated immune cells in response to known proinflammatory mediators. By considering and quantifying both morphology, proliferation, and growth rate we were able to build a full picture of macrophage function and further understand temporal cellular responses to pathogens. This may have merit in developing therapeutic interventions targeting macrophages in disease models of uncontrollable inflammation as well as being developed for broader applications in the future.
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