Using Immunofluorescent Cell Marker Quantification as an Identification Method for M 1 and M 2 Macrophage Phenotypes

Macrophages as antigen presenting cell (APC) play a vital role in orchestrating immune responses against foreign materials. The activation status of macrophages could be determined by tracking the expression of various cell markers that can be a signal for their immune activity behaviour following cellular stimulation either towards healing or inflammation. Previously numerous immunofluorescent cell markers have been used for distinguishing between pro-inflammatory macrophage phenotype (M1) and anti-inflammatory macrophage phenotype (M2) qualitatively, although most of those fluorescent cell markers express in both phenotypes. We have developed a new strategy to identify M1 and M2 phenotype quantitatively by using immunofluorescent cell markers. This approach enables the identification of different macrophage functional phenotypes quantitatively, and their degree of polarisation. Macrophages were polarised to M1 and M2 phenotypes by GM-CSF+IFN-γ and M-CSF+IL-4, respectively. Control cells were un-polarised (naïve) macrophages or monocytes were considered as macrophage progeny. For assessing cell polarisation all cell types were stained for nucleus. Also, their surface markers were stained with calprotectin for M1 cells and mannose receptor (MR) for M2 cells, followed by fluorescent microscopy examination. Cell images were analysed using CellProfiler software in order to measure the fluorescent signal intensity of the cell markers, and create a specific profile for each cell type. These profiles formed the basis for M1 and M2 phenotype identification. By using such fluorescent signal parameters we were able to identify M1 and M2 phenotypes effectively and distinguish them from naïve macrophages and monocytes.

The complexity of characterisation in M1/M2 human macrophages by surface cell markers has encouraged investigation in an alternative approach that would be less resource-intensive and simpler.
The aim of the present study was to quantify the cell surface markers signal intensity, calprotectin (M1 cell marker) and MR (M2 cell marker) in M1 and M2 macrophage subtypes respectively.Data were collected and used to build a threshold to identify different macrophages status.Monocytes were stimulated in vitro for 6 days with M1 (GM-CSF+IFN-ʏ) or M2 (M-CSF+IL-4)-inducing cytokines.acadj@garmian.edu.krdVol.5, No.2 (June, 2018) 1.

Monocyte isolation and culture
Buffy coats were obtained from the National Blood Service following Ethics committee approval (National Blood Services, Sheffield, UK; 2009/D055).Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats by Histopaque-1077 (Sigma-Aldrich) density gradient centrifugation.Monocytes were isolated from PBMCs using the MACS magnetic cell separation system (positive selection with CD14 MicroBeads and LS columns, Miltenyi Biotec) (Rostam et al., 2017, Rostam et al., 2016, Singh et al., 2017).RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine and 100 U/ml penicillin and 100 µg/ml streptomycin (all from Sigma-Aldrich) (henceforth referred to as "complete RPMI medium") used for monocytes were suspension with the cell density of 1 x 10 6 cells/ml.1 ml of the suspension (=1 x 10 6 monocytes) was seeded on round coverslip 12mm in each well of 24 tissue culture well plate, then incubated at 37˚C, 5% CO 2 in a humidified incubator for six days.
Cells of different activation states were produced using cytokine addition monocytes seeded on normal glass slides: for polarisation to M1 a mixture of 20 ng/ml IFN-γ (R&D Systems) and 50 ng/ml GM-CSF (Miltenyi Biotec) was added to a total volume of 1ml; for M2 differentiation 20 ng/ml of IL-4 (Miltenyi Biotec) and 50 ng/ml M-CSF (Miltenyi Biotec) were added to the well volume of 1 ml.The cells were incubated at 37ºC, 5% CO 2 in a humidified incubator for 6 days.On day 3 of incubation, 500 µl of the medium was replaced with fresh complete RPMI medium containing the same concentration and mix of cytokines that were used for cell stimulation at the beginning of culture.After six day of incubation M1 and M2 macrophages were stained with calprotectin (M1 marker) and MR (M2 marker).Images of both phenotypes were taken with an automated fluorescent microscope.Automated image analysis software (CellProfiler) was used to measure and record the maximum fluorophore intensity per image for nine different images.This was repeated for two different samples for the same biological donor and the values of calprotectin in M2 and MR in M1 were calculated.These values were used as threshold intensity values of calprotectin and MR in order to categorise cells not exposed to cytokines, with each cell exhibiting fluorescence intensity above these calprotectin and MR thresholds categorised as M1 and M2 respectively.

Polymer Surfaces Synthesis
Polymer surface were been synthesized by using methods described previously (Anderson et al., 2004, Hook et al., 2012).Briefly, Each polymerisation solution was composed of monomer (50%, v/v) in dimethylformamide with photoinitiator 2,2dimethoxy-2-phenyl acetophenone (1%, w/v).Polymers were purchased from Aldrich, Scientific Polymers and Polysciences and coated onto epoxy-coated slides (Xenopore) dip-coated with poly(2-hydroxyethyl methacrylate) pHEMA (4% w/v, Sigma) in ethanol (95% v/v in water).Coated surfaces were sterilised by exposure to UV light for 15 minutes.The hits materials were scaled up as polymer coupons formed by pipetting polymerization solution (6µL) onto a pHEMA coated slide and irradiating for 10 mins at O 2 < 1300 ppm with a long wavelength UV source.Once formed, volatile components were removed from the polymers at <50 mTorr for 7 days.Polymers were characterized by water contact angle measurements and time-of-flight secondary ion mass spectrometry as described previously (Taylor et al., 2007, Urquhart et al., 2007).

Determining macrophage pro or anti-inflammatory phenotype using fluorescent microscopy
The expression of cell surface marker use as a method to identified macrophage.Automated microscopy with high quality image analyser was used to perform high throughput scanning for the adhered cells on glass slide surface (Murray et al., 2014, Xue et al., 2014).High throughput method was used for the glass slide screening by the automated fluorescent microscopy (IMSTAR) to determine the level of calprotectin (M1 marker) expression in M1 pro-inflammatory macrophages population, and the level of MR (M2 marker) in M2 anti-inflammatory macrophage population (Rostam et al., 2016) (Figure.1).To examine macrophage polarisation under the effect of polymers, we examined the expression of markers in both populations (cytokine polarised, M1 and M2) on glass slides.The mean of fluorescent intensity pixel for calprotectin and MR value was calculated in M2, M1 cytokine polarised cell, respectively.That was used to categorised the phenotypes of seeded macrophages on the polymers surfaces as M2 or M1 when they their marker fluorescent expression above the average levels in M1 or M2 respectively.

Macrophage polarisation
Using fluorescence microscopy the number of MR + and calprotectin + cells was quantified for each using the M1 and M2 identification criteria developed on cytokine differentiated naïve macrophages.Homo-polymer number (decyl methacrylate) was the most effective at polarising the cells towards the M2 phenotype, with 2.9 times more cells expressing MR (68±28 cells) compared to calprotectin (24±21cells) as seen in Figure 2. A high degree of cell attachment (88±23 cells) was also observed on this homopolymer.Other materials polarising cells towards the M2 phenotype included homopolymer numbers (hexyl acrylate) with ratios of MR + to calprotectin + cells of 2.6 The homo-polymers(hydroxypropyl acrylate) were the most effective at polarising cells towards the M1 phenotype whilst still supporting the attachment of more than 50 cells , with 3 times more calprotectin + cells than MR + cells (Figure 2).

Discussion
In this work, for first time MR and calprotectin has been quantified in M1 and M2 macrophage subsets, and used as threshold for M1 and M2 identification.M2 macrophages expressing high amount of MR can be induced in vitro by IL-4 and IL-13 (De Paoli et al., 2014).MR in macrophages observed to operate control of innate immunity and it is also believed to be involved in regulating antigen presentation and lymphocytes trafficking to lymph nodes.In addition, MR as a scavenger receptor has preference for collagens and for glycosylated proteins (Hagert et al., 2018).However, M2 macrophages may express low amount of calprotectin (Hsu et al., 2009) which has acadj@garmian.edu.krdVol.5, No.2 (June, 2018) been considered as M1 cell marker (Bartneck et al., 2010, Rostam et al., 2017, Rostam et al., 2016).M1 macrophages which can be stimulated by Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) (Huang et al., 2017) can be characterised by expression of a high level of calprotectin (Bartneck et al., 2010, Rostam et al., 2017, Rostam et al., 2016) which could induce pro-inflammatory cytokine production properties of the macrophages.However, calprotectin may express in monocytes and M2 macrophages (Xia et al., 2018).
From visual inspection of cell surface marker expression of macrophages, immediate differences in their respective signals has been noticed, these differences has been quantified by CellProfiller software which can detect signal intensity of each cell markers and their distributions across the cell surface (Rostam et al., 2017).The mean of maximum expression of calprotectin in M2 phenotype cells can be used as a save threshold for M1 phenotypes.In addition, M2 can be identified when the mean of MR signals of any cell exceeded the mean of maximum MR intensity signals of M1 phenotypes.Later, this way of cell identification successfully has been used to identify the macrophage polarisation to word M1 and M2 under the impact of surface chemistry modulation (Rostam et al., 2016).Biomaterial surface chemistry has previously been shown to modulate macrophage adhesion and function (Rostam et al., 2015).In this study a high throughput screening strategy has been used to collect image data from incubated cells, the M1 and M2 phenotypes controls, and from macrophages seeded on different homo-polymers.This new approach used to investigate the effect of different homo-polymers surface chemistries on human monocyte differentiation.By using the method M1 and M2 biased homo-polymers has been identified depending on data analysis by the new method effectively.

Conclusion
Using immunofluorescent cell marker quantification, as a new effective identification method for M1 and M2 macrophage phenotypes in mixed macrophage population could pave the way for further investigations in this area.This method is capable of achieving high degrees of accuracies, in contrary to macrophage phenotype heterogeneity that can affect the cell marker signal expression.However, presented data provide strong indications for ability of this method to perform M1 and M2 subtype identification with less resource intensive and fast way of identification, still it is be too early to suggest this approach as an alternative for conventional cell phenotyping in wide scale cell identification.

Figure 2 :Figure 2 :
Figure 2: Determining macrophage pro or anti-inflammatory phenotype using fluorescence microscopy (A,B) Scatter plot for number of M2 /M1polarised cells with cytokines on glass slide, X-axis average total cell number of the adherent cells ,Y-axis is number of cells expressed MR + (M2-phenotype)/ number of cell expressed calprotectin (M1phenotype)on glass slide .n=3(D, homo-polymer arrays experiment) and 2 (E, co-polymer arrays experiment) of different samples (M1 and M2) for each sample with 2 replicates.