Analyse métabolomique par RMN de cellules THP-1

L’article, Apoptosis and inflammation-related metabolic alterations in monocytic cells

: comparative approaches by liquid 1H-NMR and HR-MAS on intact cells fait l’objet d’une soumission dans la revue Pathologie Biologie en 2010. Le modèle choisi, une lignée de monocyte THP-1 à basse densité (en termes de nombre de cellules), a été soumis à une induction d’apoptose ou d’inflammation en utilisant soit le 7-kétocholestérol, soit des lipopolysaccharides (LPS).

Nous avons réalisé une comparaison de trois méthodes de préparation et d’enregistrement des données RMN dans l’accès aux données du métabolisme cellulaire. Les deux méthodes d’analyse effectuées par RMN en milieu liquide se différencient par l’existence ou non d’une extraction versus l’utilisation de lyophilisations, par opposition aux techniques spécifiques de HR-MAS. En considérant à la fois la quantité de matériel cellulaire requise, le seuil de détection, la résolution des spectres obtenus et les contraintes techniques (en premier lieu le temps d’acquisition), ainsi que la fiabilité des données, il est apparu que ces trois méthodes offrent des avantages et des limites différents, c'est-à-dire in fine un champ d’application complémentaire. Ainsi, bien que les trois méthodes donnent des résultats comparables, les différences modérées observées en particulier entre HR-MAS et extraits PCA apportent certaines limitations.

L’extraction par lyophilisation parait préférable avec de petites quantités cellulaires. Dans ce cas, les pertes sont réduites du fait de l’absence de traitement, et les informations peuvent être obtenues sans dégradation majeure au prix d’acquisitions plus longues.

Seules les méthodes HR-MAS optimisées donnent accès à l’étude de cellules vivantes, et offrent la possibilité d’une étude simultanée des métabolites solubles et des lipides membranaires. Les contraintes techniques de contrôle de survie cellulaire, de température et de rapidité d’acquisition apportent cependant une certaine limitation.

Finalement, l’utilisation des extraits perchloriques « classiques » est à réserver pour des échantillons à forte densité cellulaire, compte tenu de la perte occasionnée inévitablement par le processus d’extraction. La non reproductibilité entre extraits successifs peut également constituer une limite significative à la méthode.

Dans l’optique de l’étude des MAP Kinases, cette étude préliminaire a conduit à sélectionner, compte tenu du modèle cellulaire et des résultats attendus, les techniques de HR-MAS et des méthodes de traitement des données associées (statistiques, JMRUI…).

Apoptosis and inflammation-related metabolic alterations in monocytic cells : comparative approaches by liquid ¹H-NMR and HR-MAS on intact

cells.

Comparaison de trois méthodes RMN pour l’analyse des altérations métabolites liées a l’apoptose et l’inflammation.

S. Follot ^a,*, D. Crouzier ^a, C. Delaunay ^b, T. Poyot ^c andJ.-C. Debouzy ^a

a

IRBA/CRSSA, Unité de Biophysique, BP82, 38702 La Tronche cedex, France. b

IRBA/CRSSA, Unité de Radiohématologie Expérimentale, BP82, 38702 La Tronche cedex, France.

c

CRSSA, Unité de Radiobiologie, BP82, 38702 La Tronche cedex, France.

* Corresponding author Fax : 33-(0)4-76-63-69-22 e-mail : sfollot@crssa.net

Abstract: Three NMR methods were used to assess the metabolic changes occurring after 7-ketocholesterol or lipopolysaccharide-induced apoptosis and inflammation in cell samples of low density. The samples were analysed by recording classical liquid ¹H-NMR on perchloric extracts or after lyophilisation of the sample, and also by using HR-MAS NMR method on intact cells. It was found that the relative contribution of NMR-visible lipids and soluble metabolites resulted in quantitative differences in relative metabolites amounts in the different samples. The all experiments showed a significant decrease of myo-inositol after apoptosis induction. Besides, when sufficient amounts (250,000,000) of cells were available, PCA extracts gave the NMR spectra with the best resolution, ie with no lipid contribution. By way of contrast, whereas lyophilisate only required limited number of cells, the spectrum analysis was uneasy to analyse, due to partial superimposition of lipid contributions compared with PCA extracts. Finally, HR-MAS NMR of intact cells was found best method by allowing the simultaneous study of soluble metabolites and partial quantitative approach of relative lipid contributions.

Keywords: Apoptosis ; Monocyte ; Metabolic ; Inflammatory ; Nuclear Magnetic Resonance.

Résumé: Les changements métaboliques de cellule THP-1 après activation par le 7-ketocholesterol ou par des lipopolysaccharides ont été étudiés par trois méthodes RMN. Les échantillons ont été analysés par RMN liquide sur des extraits perchlorique (PCA) ou après lyophilisation, mais aussi par RMN HR-MAS. Toutes les méthodes ont montré une diminution significative de myo-inositol après l'induction de l'apoptose. Les expériences ont été réalisées avec une faible quantité de cellule (15.000.000) et une plus grande quantité de cellule (250.000.000). Seule l’extraction PCA donne de meilleur résultat avec une plus grande quantité de cellule. La lyophilisation permet une analyse des changements métaboliques sur une faible quantité de cellule, mais la technique est limitée par la présence de lipide qui varie suivant l’échantillon. La HR-MAS est la seule méthode qui permet l’analyse de cellule intacte. Elle permet aussi l’analyse sur le même spectre des métabolismes et des lipides grâce au rapport CH2/CH3.

Mots clés: Apoptose ; Monocyte ; Metabolisme ; Inflammation ; Résonance Magnétique

Introduction

The knowledge of cell metabolism regulation and perturbation in the presence of external or intrinsic aggressions has been for long a challenge[1-2]. Up to recently, this was mostly achieved by the way of indirect methods (fluorescence[3], radio-[4] or GC-MS[5], morphologic observations[6] …) or by performing the biochemical analysis of percholric extracts for soluble metabolites[7-8] or also lipid extracts[9-11]. In both cases, NMR analyses were performed by high resolution NMR methods in solution. By way of contrast, HR-MAS NMR (magical angle spinning high resolution NMR) requires no sample preparation and thus have been increasingly been used for the analysis of solid of viscous biological samples such as biopsies or natural membranes[12-13]. More recent studies dealt with cells cultures[14], frequently with high density of cells[15]. However, no direct comparisons of metabolic changes have been, up to now, performed in diluted samples, between solution studies and HR-MAS[16]. This paper presents an attempt for such a comparison. Besides the technical aspect, a reference model both of cell system and of classical metabolic stimuli were required. THP-1 monocytic cell lines are a classical model for activation, which growth and survival tests are easy to perform[17], and activation obtained by various reagents such as surfactant protein A[18].

Cell responses to extra cellular stimuli involve many interactive metabolic pathways to achieve the putative functions present in an organism such as cell proliferation or differentiation (cell cycle)[19], inflammation, embryogenesis, or cell death[20-21]. These pathways are well identified in THP-1 lines.

Usually, such responses are at least partially mediated by phosphatase or kinase enzymatic proteins[22-23]. More precisely, cascades of intracellular signalling and regulations occur by the way of mitogen activated kinases (MAP). Among them, the P38 subgroup of kinases, while sharing many similarities with the other kinases subgroups, it is a convenient tool for testing the dramatic metabolic changes observed after their stimulation. Moreover, P38 can be easily activated by several different ways[24] e.g external signalling such as LPS (lipopolysaccharide, a bacterial toxin)[25-26], or other cells components like 7-ketocholesterol (7-KETO), a cholesterol derivative identified in atherosclerotic plaques[27], the latter acting via membrane integration and cholesterol metabolism perturbation. It is well known that LPS, a constituent of the outer membrane of Gram-negative bacteria, activates

macrophages (THP-1[28]) strongly and induces production of a number of molecules including cytokines, eicosanoids, and free radicals. It thus participates in various events associated with the inflammatory while the 7-KETO induced cell apoptosis with cell cycle arrest[29]. These reagents were thus selected under the scope to obtain and compare metabolic informations about lipidic and soluble molecules.

One of the major analytical challenges of metabolomic studies by NMR methods is the ability to measure and quantify metabolites of low concentration. Classical methods, such as the ¹ H-NMR spectroscopy, HR-MAS and GC-MS can not provide unequivocal quantification and identification even for the simplest metabolomic dataset. It was thus of interest to identify the most powerful analytic method to enable the study of low-density cells samples. To address this issue, different NMR approaches (HR-MAS, liquid NMR after lyophylisation or extraction) for metabolic profiling of THP-1 cells samples were used. Two distinct THP-1 cells activation mechanisms were used (LPS, 7-KETO) and metabolomic consequences then analysed.

Materials and methods

Cell culture and viability test

The human myelomonocytic cell line, THP-1 cell line (ECACC 88081201; Sophia-Antipolis, France) was used. These non-adherent cells were grown in suspension in RPMI 1640 with glutamaxTM-I (Gibco, Eragny, France) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin) (Gibco), supplemented with 10%(v/v) heat-inactivated fetal calf serum (Gibco). The THP-1 cells were incubated at 37°C under a 5% CO2/95% air atmosphere (v/v). For all experiments, the monocytes were stimulated with optimal dose (0.1-10 µg/ml) of bacterial LPS (Escherichia coli serotype O111:B4) and 7-KETO (10-40 µg/ml) were obtained from Sigma (la Verpillère, France).

The trypan blue exclusion test was used to assess cell viability. The cell suspension was well mixed with an equal amount of 1% trypan blue solution in PBS. The solution was introduced in the Malassez cells (microscope). The cells viable were round shaped and bright, while dead cells looked completely blue due to the incorporation of trypan through the damaged membranes. The viability test was performed immediately for preparation of cell suspension in the rotor and immediately after the removal from the rotor itself.

Flow cytometry

The cell suspension was collected and centrifuged, and the supernatant was discarded. The pellet was recultivated in PBS to 5.10⁶ cell/ml. Cells were fixed with 0.5 ml ethanol (-20°C) over night. Cells were washed, and resuspended at 106 cells/ml. 1 µl of propidium iodide (0.5 mg/ml) and 60 µl RNase A (500 I/mL) were added. The solution was incubated 20 minutes. The samples were stored at 4°C until analysis by flow cytometry using LSR 2. Afterward, 10.000 cells were acquired, and the cell cycle was analyzed. The laser configurations on these instruments allow analysis of cell samples stained with 488 nm, 633 nm.

Perchloric acid extraction and lyophilisation conditions

The cultures washed twice with cold PBS, immediately frozen in liquid nitrogen and stored at –80 °C for further treatment. Perchloric acid (PCA) extraction was performed following

standard procedures[30]. Briefly, the semi-dry cell paste was resuspended in 0.3 ml of 12% PCA. After homogenisation, the final cell suspension was centrifuged at 8000 g for 10 min and the supernatant neutralised to pH 7.4 with KOH. The sample was again centrifuged at 8000g for 10 min, the supernatant was lyophilised, and the dry residue dissolved in 0.5 ml of D₂O for NMR analysis.

The cultures washed twice with cold PBS, immediately frozen in liquid nitrogen and stored at –80 °C for further treatment. The cell sample was lyophilized prior to use in order to obtain a dry surfactant powder for preparations of starting solutions, using a Heto–Holten freeze dryer (Heto Drywinner 3). The dry residue dissolved in 0.5 ml of D₂O f and placed in a 5mm NMR tube to NMR analysis.

NMR experiments

All HR-MAS experiments were performed on Bruker AVANCE-DMX-400 (proton frequency 400.13 MHz) using a Bruker HR-MAS probe head. Samples were loaded in zirconium oxide 4mm cylindrical rotor with spherical insert (internal volume of 50 µl). Samples were spun at 1900 Hz.

The 15 x 10⁶ cells in culture solution were collected at discrete times and centrifuged for 5 min at 400g to collect the THP-1. The cell pellet was washed three times with PBS D2O and a finally centrifugation. The half-dry cell paste was resuspended in 30 µl of a PBS D₂O TSP (3 mM Trimethylsilyl-2,2,3,3-tetradeuteropropionic acid sodium salt). The solution was rapidly placed in rotors with spherical insert. The rotors were immediately transferred into the HR-MA probe and acquisition started after 3 min of temperature equilibrium.

1

H-NMR experiments were recorded 16 K digitization points. A Carr-Purcell-Meiboom-Gill (CPMG) synchronized pulse sequence with an echo-time of 30 msec was used to reduce the lipid contributions[31]. The interpulse delay was 345 µsec and water was presaturated during the relaxation delay of 2 s, 12 min were necessary for the acquisition of 256 transients.

High-resolution proton nuclear magnetic resonance studies were performed at room temperature on a Bruker AVANCE-DMX-400 (proton larmor frequency 400.13 MHz) equipped with the BBI-Z-gradient probe. Spectral parameters using 4000 Hz spectral width, 32 K digitization points, a recycling delay of 2 s and presaturation of the solvent resonances

Metabolite quantification

Quantification was performed with jMRUI software package using the “subtract-QUEST” procedure[32]. The procedure uses a simulated database set including 14 metabolites: acetate (Ace), aspartate (Asp), alanine (Ala), choline (Cho), creatine (Cr), glutamate (Glu), glutathione (Gsh), glycine (Gly), glutamine (Gln), lactate (Lac), myo-inositol (M-Ins), phosphocholine (Pc), valin (Val) and taurine (Tau). The amplitude of metabolites calculated by QUEST was normalized to the total spectrum signal and then only relative concentrations were produced.

Statistical analysis

Statistical analysis of the data was done by an analysis of a non-parametric test for assessing whether two independent samples of observations come from the same distribution (Statistica® 7.1 software, Statsoft Inc., Tulsa, OK, USA). The Mann–Whitney U test was used to determine statistical significance. The experiments were reproducibly repeated five times at least and ten times maximum. The significance level was set at p < 0.05.

To perform this study, the first step was to set up the experimental conditions, and more precisely to know the amounts of LPS or 7-KETO to use, by regard of cell survival; this was realized in the first section.

Results.

Preliminary set-up and controls : Cell survival and 7KS, LPS concentration determination.

The results are presented on the figure 1. Equal amounts of cells (I.E. 15.10⁶ cells per sample) were firstly incubated for 0, 1, 6, 24 hours in the presence of 7-KETO (10-40µg/ml) or LPS (0.1-10µg/ml), according with the methods classically described in the literature[33-34]. Thus, the initial plot of cell death percentage, drawn from trypan blue experiment was not equal to zero, while being extremely close between the different samples. In the presence of LPS, no further evolution was observed for 6 hours; conversely, a slight decrease in death rate was observed at 10µg/mL for later times. Such a feature is generally related to an inhibition of the activation by LPS occurring after initial inflammation step[35]. As this later evolution was not present at 0.1µ g/mL while and similar as that of control cells, this LPS concentration was used in the followings experiments. Such was not the case for 7-KETO samples. As expected progressive cells death was obtained down to 75% and 40% for 40 and 10µ g/mL, respectively. Since the former concentration induced too dramatic effects, the concentration of 10µ L/mL was used in the following experiments.

A control of the mechanisms involved was also performed by flow cytometry counts. As shown on the table I, absolute and time dependant growth phase distribution of the cells in the presence of LPS were found quite similar as the control sample (i.e. around 54 to 50%in G0/G1, 30-34% in S and 16 to 15% in G2 phase from 1 to 6hours). Conversely, a dramatic reduction of cell population in S phase associated with an increase of cells stopped in G0/G1 revealed that apoptosis had occurred as expected in this case.

This allowed us to undertake metabolic comparisons between the different preparations and NMR methods, as described in the next sections.

NMR spectra and metabolite selection.

The figure 2 shows typical ¹H-NMR spectra obtained from the three preparations. The observation of some resolved lines (less than 1Hz) confirmed the presence of soluble metabolites which assignment could be derived from the literature[36-37] and ²D-NMR ¹ H-experiments (COSY, TOCSY, HMQC, HMBC). These different species are presented on the

table 2. Other broader lines (15-25Hz) were also detected and identified as lipidic resonances

from similar experiments and classical data[38], e.g. terminal methyl groups and methylenic chain resonances found around 0.8 and 1.6ppm, respectively.

Figure 1: Plot of percentage of cell death at 0, 1, 6 and 24 hours as THP-1 (–♦–), after activation by LPS at 0.1µg/ml (–

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Table 1: Effect of LPS and 7-KETO on cell cycle progression (%) in THP-1 macrophage. control 7-keto lps time (hours) _{G0/G1 S G2 G0/G1 S G2} G0/G1 S G2 0 54,9 ± 1,2 29,0 ± 1,1 16,1 ± 1,9 53,9 ± 1,5 30,2 ± 1,4 15,9 ± 1,3 53,6 ± 1,8 30,1 ± 1,8 16,3 ± 0,8 1 54,2 ± 1,5 29,9 ± 1,3 15,9 ± 1,5 54,1 ± 1,8 30,8 ± 1,2 15,1 ± 1,2 53,4 ± 1,7 31,2 ± 1,1 15,4 ± 1,4 6 50,8 ± 1,7 34,0 ± 1,4 15,2 ± 1,2 55,7 ± 2,2 29,3 ± 1,3 15 ± 1,3 51,4 ± 2,0 33,9 ± 1,3 14,7 ± 1,0 24 48,5 ± 1,4 36,8 ± 1,1 14,7 ± 1,7 62,2 ± 2,5 23,0 ± 1,2 14,8 ± 1,5 50,7 ± 2,1 34,4 ± 1,6 14,9 ± 1,6

Figure 2: A) Typical ¹H-NMR spectrum of metabolites extracted by PCA from cells samples; B)1

H-NMR spectrum of metabolites extracted by lyophilisation from cells samples;

C)1

Due to peak superimpositions, numerous resonances could not be clearly analysed and were thus discarded from the rest of the study.

Spectra of perchloric extracts (PCA). As expected from this method, no contribution of lipid protons was detected, thus resulting in well-resolved spectra. However, the spectrum presented on the figure2-A exhibits a poor signal to noise ratio (under the same acquisition

conditions), indicating signal intensity and corresponding metabolite concentrations losses had occurred as a consequence of the extraction procedure used[39]. Thus only several lines could be identified, despite the very best resolution (a signal to noise ratio comparable with the other methods required 250 10⁶ cells). Acetate (Ace) and myo-inositol (M-Ins) time course are presented on the figure 3. After an initial shot down of all the contributions, a significant

increase was observed later for Ace in the presence of LPS (0.1µ g/mL), while M-Ins remained low. In the presence of 7-KETO (10µ L/mL), a simultaneous drop of Ace and M-Ins was found after 1 hour incubation, indicating, as published elsewhere[40], that apoptosis had well occurred.

Lyophylisates. (Figure 4).This method induces a insignificant material loss: hence, a good

signal to noise was obtained, and many compounds could be identified on the NMR spectra (Figure 2-B). Since no extraction had been performed, cell lipids resonances were still

detected despite the NMR echo sequence used, even if lipidic molecules had been partially removed by the centrifugation. However, this minor contribution of lipid protons was sufficient to overjammed the spectrum. Finally, in LPS (0.1µg/mL) experiments, one could still identify the two-step evolution of Ace contribution (PCA experiments). A significant increase of Gln was observed at 1 and 6 hours incubation. Also, a biphasic evolution of Gsh was found.

In the presence of 7-KETO (10µ L/mL), a similar evolution was noticed for M-Ins and Ace that in PCA experiments. Also, significant increase of Val and Gly were observed to 24 hours incubation.

Besides, lipid contributions were too overlapped to be reasonably exploitable, what create a variation in handling data.

HR-MAS. On the figure 2-C one can notice that the resolved lines of soluble molecules

coexist with several lipidic contributions. However, due to the MAS methods, their contributions were reduced in intensity, and exhibited a better resolution. This allowed to

Figure 3: Plot of percentage metabolites of control value determined by quantization of ¹ H-NMR spectrum of cells extract by PCA at 0, 1, 6 and 24 hours; A) After activation by LPS quantization of Ace (●) and M-Ins (■); B) After activation by 7-KETO quantization of Ace (●) and M-Ins (■); indicates significant test with p<0.05.

Figure 4: Plot of percentage metabolites of control value determined by quantization of ¹ H-NMR spectrum of cells extract by lyophilisation at 0, 1, 6 and 24 hours; A) After activation by LPS quantization of Ace (

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investigate further these lipidic resonances. The figure 6 shows the time course curves built

from the ratio of methylenic and methyle contributions. One can here notice an initial jump upon LPS (0.1µ g/mL) addition, followed by a back evolution down to the initial level. 7-KETO (10µ L/mL) curves only exhibited continuous decrease in this ratio, indicating that membrane damages still occur even at late times of evolution (Figure 5).

The localization and quantization of Ace by HR-MAS was precluded by overjamming of lipidic resonances giving unreproducible results. In LPS experiments, the increase of GLy at 24 hours could be related with activation of inflammation pathway. In the presence of 7-KETO, M-Ins, Gly and Gsh evolution was found as in lyophylisation experiments. Tau concentrations were also lower in apoptosis cells at 24 h.

Figure 5: Plot of percentage metabolites of control value determined by quantitation of ¹ H-MAS NMR spectrum of intact cells at 0, 1, 6 and 24 hours; A) After activation by LPS quantitation of Gly (■); B) After activation by 7-KETO quantitation of Tau (*), Gly (+), Gsh (■) and M-Ins (▲); indicates significant test with p<0.05.

Figure 6: Plot of ratio CH2/CH3 determined by deconvolution (The signal was estimated using ACDLABS 10.0 software) spectrum of intact cells at 0, 1, 6 and 24 hours as THP-1 (–

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