Ahmed Al-Kattan
email: ahmed.alkattan@ensiacet.fr

Pascal Dufour

Jeannette Dexpert-Ghys

Christophe Drouet
email: christophe.drouet@ensiacet.fr

Preparation and Physicochemical Characteristics of Luminescent Apatite-Based Colloids

Luminescent colloidal nanosystems based on europium-doped biomimetic apatite were prepared and investigated. The colloids were synthesized by soft chemistry in the presence of a phospholipid moiety, 2-aminoethylphosphoric acid (AEP), with varying europium doping rates. Physicochemical features, including compositional, structural, morphological, and luminescence properties, were examined. Experimental evidence showed that suspensions prepared from an initial Eu/(Eu + Ca) molar ratio up to 2% consisted of singlephased biomimetic apatite nanocrystals covered with AEP molecules. The mean particle size was found to depend closely on the AEP content, enabling the production of apatite colloids with a controlled size down to ca. 30 nm. The colloids showed luminescence properties typical of europium-doped systems with narrow emission bands and long luminescence lifetimes of the order to the millisecond, and the data suggested the location of Eu 3+ ions in a common crystallographic environment for all the colloids. These systems, stable over time and capable of being excited in close-to-visible or visible light domains, may raise interest in the future in the field of medical imaging.

Introduction

Lanthanide-based phosphors have been used for many years in the field of optical devices (lasers, phosphors in color TV tubes, etc.) [START_REF] Go ¨rller-Warlrand | Handbook on the Physics and Chemistry of Rare Earths[END_REF][START_REF] Franz | In Ullmann's Encyclopedia of Industrial Chemistry[END_REF][START_REF] Heyes | [END_REF] and, more recently, for medical applications. [4][5][6][7] In particular, the development of luminescent probes in biology faces the issue of particle size, 8 especially when interactions with cells are foreseen, either in vitro or in vivo. [9][10][11][12][13][14][15][16][17] Organic dyes, such as fluoresceine, green fluorescent protein, or rhodamine, are fluorescent probes commonly used in biology. However, these molecules do not allow an extended analysis of biological tissues due to fast damage under irradiation, as well as broad spectrum profiles and low photobleaching thresholds. To remedy this shortcoming, new classes of labeling agents, such as semiconductor quantum dots (e.g., CdSe-CdS, CdSe-ZnS, InAs), silica, or phosphate nanoparticles, were introduced more recently. [18][19][20][21] In particular, semiconductor systems show a higher photostability than usual organic fluorophores and a lower spectral width of emission, and their color can be adequately selected by modifying the particle size. These aspects have led many research groups to use quantum dots in biological assays. 20,21 However, the luminescence of these systems is accompanied by a flickering effect, leading to random appearance and disappearance of the luminescence, which limits the spatial resolution during bioluminescent analyses. 22,23 Also, semiconductors appear as rather toxic systems 24,25 due to the presence in their composition of Cd or As elements, for example.

Alternative routes for the preparation of other types of luminescent probes for a potential use in biology are still being explored. Lanthanide-doped inorganic nanoparticles have, for example, several advantages compared to quantum dots: their fluorescence is characterized by narrow emission bandwidths, and they show a high photochemical stability and a long fluorescence lifetime. Also, as for quantum dots, different colors are available by varying the luminescent center used (e.g., Tb, Eu, etc.). Increasing efforts have been focused on the production of luminescent nanoparticles doped with lanthanide(III) ions, such as lanthanum fluoride 26 and lanthanum phosphate, [27][28][29][30] or yttrium vanadate. 31,32 Favorable biological properties of such materials were reported in the last years. 18,33 Nanocrystalline calcium phosphate apatites analogous to bone mineral, Ca 10-x (PO 4 ) 6-x (HPO 4 ) x (OH) 2-x (0 e x e 2), doped with lanthanide ions, such as Eu 3+ , or Tb 3+ have also been prepared. [33][34][35][START_REF] Fanjul | In Vitro Cell[END_REF] The physicochemical characteristics and surface state of biomimetic apatites have largely been investigated, especially in view of bone regeneration applications. 37 Also, the ability of the apatite structure to accommodate many types of ionic substituents has been widely reported. 38,39 Some luminescence properties of Eu 3+ -doped apatites were examined in several studies, [40][41][42][43][44] unveiling the possibility to obtain a red fluorescent probe. Moreover, the possibility to excite this ion at low energy (in or near the visible domain) may prove to be suitable for studying biological tissues.

Recently, 35 colloidal suspensions of apatite nanocrystals have been prepared in our laboratory for the setup of nanophosphors. However, the synthesis protocol used was based on precipitation from phosphoric acid and freshly calcined calcium oxide (under an inert atmosphere for avoiding the rehydration of CaO) and was thus hardly transposable to an industrial scale. The preparation of nonluminescent apatite colloids was then recently revisited in detail, revealing the possibility to obtain apatite colloids with controlled particle size, starting from nontoxic easily handled ionic salts. 45 In the present work, we succeeded in preparing stable luminescent apatite colloids doped with a variable Eu/(Eu + Ca) molar ratio, starting from easily handled innocuous starting salts. The physicochemical characteristics of these colloids, including, in particular, the size, morphology, composition, and luminescence properties, were explored based on several complementary techniques.

Materials and Methods

2.1. Preparation of Apatite Colloids Doped with Europium. In this work, the preparation of colloidal apatite suspensions was based on the rapid coprecipitation at room temperature of calcium, europium, and phosphate salts. The synthesis was run in deionized water in the presence of a biocompatible stabilizing agent: 2-aminoethylphosphoric acid, "AEP" (under the form NH 3

+ -CH 2 -CH 2 -O-P(O)(O -) 2
at pH 9.5). After precipitation, the samples were aged at 100 °C for 16 h. AEP, a natural phospholipid moiety, was shown in previous studies to interact strongly with calcium ions [45][46][47] while providing an electrosteric repulsive effect, limiting apatite crystal agglomeration. Unless otherwise specified, the starting AEP/ (Ca + Eu) molar ratio was set to 1. This value has been shown 45 to lead to stable deagglomerated apatite particles with a mean particle size around 30 nm. The effect of AEP content was also followed. To this purpose, some suspensions were prepared starting from a AEP/(Ca + Eu) ratio of 0.8, 0.6, 0.4, and 0.2.

For each preparation, three solutions were prepared. Solution (A) contained a total of 4.87 mmol of calcium nitrate (Ca(NO 3 ) 2 • 4H 2 O) and europium nitrate (Eu(NO 3 ) 3 • 6H 2 O) with a variable Eu/(Eu + Ca) molar ratio in the initial reaction mixture (0, 1.5, 2, 4, 7, 10, and 100%), dissolved in deionized water. Solution (B) contained 4.87 mmol of AEP in deionized water. Finally, solution (C) was prepared by dissolving 1.62 mmol of di-ammonium hydrogen phosphate in deionized water, with an initial molar ratio (Ca + Eu)/P of 0.33. This ratio was used previously for the preparation of apatite colloids in the presence of AEP. 45 In a typical procedure, solution (A) was mixed with solution (B) under constant stirring. The acidic pH of the resulting solution, referred to as solution (D), as well as that of solution (C) was adjusted to 9.5 by addition of ammonia. Such an alkaline pH value was selected in order to favor the precipitation of an apatite phase close to stoichiometry, which exhibits good chemical stability. 45 The pH of the colloids can be adjusted after synthesis to a physiological value prior to biological applications. The transparent colloids obtained after aging were purified by dialysis in water to remove unreacted salts (cellulose membrane, cutoff 6000-8000 Da). Ultracentrifugation at 50 000 rounds/ min, and with 3 successive washing steps in deionized water, was also occasionally used as an alternative purification process for cross-checking. No differences were evidenced between ultracentrifuged and dialyzed samples.

For comparative purposes, "reference" noncolloidal apatite suspensions were also synthesized using a similar protocol as above, but without addition of AEP. In this case, solution (B) contained only deionized water.

In all cases, a part of the suspensions was freeze-dried in view of the physicochemical powder characterization.

2.2. Physicochemical Characterization. Calcium and europium contents were determined by induced coupled plasma atomic emission spectroscopy, ICP-AES (relative uncertainty ) 3%). The amount of mineral phosphate (from apatite) in the samples was determined by colorimetry at λ ) 460 nm, using the yellow phospho-vanado-molybdenum complex 48 (relative uncertainty ) 0.5%). The AEP content in the stable colloids was drawn from nitrogen microanalysis (relative uncertainty ) 0.4%).

The crystallographic structure of the powders obtained for varying europium doping rates and after freeze-drying was investigated by way of X-ray diffraction (XRD) using a CPS 120 INEL diffractometer with the KR1 cobalt radiation (λ ) 1.78892 Å).

Fourier transform infrared (FTIR) analyses were performed on a Nicolet 5700 spectrometer, in the wavenumber range of 400-4000 cm -1 with a resolution of 4 cm -1 . Raman spectra were recorded on a Jobin Yvon HR 800 spectrometer, with a laser excitation wavelength of 632.8 nm.

The size and morphology of the apatite nanocrystals were followed by transmission electron microscopy (TEM) on a JEOL JEM-1011 set at 100 keV. The experiments were conducted on the purified colloidal suspensions. For these TEM observations, the suspensions were briefly sonicated (50 kHz, 30 s) and the carbon-coated copper TEM grids were rapidly dipped into the suspension and allowed to dry prior to analysis.

The particle size (hydrodynamic diameter, D h ) of the colloids prepared was determined by dynamic light scattering (DLS) with a Nanosizer ZS apparatus from Malvern Instruments (λ ) 630 nm). The dispersion of the data points is estimated to 0.5%. These experiments were carried out on nondialyzed suspensions so as to provide high ionic strength, giving D h values close to geometrical particle sizes and thus leading to fully exploitable DLS measurements. Punctual zeta potential measurements were also made, as mentioned in the text, using this apparatus.

The luminescence properties of the colloids were investigated using a Horiba Jobin Yvon Fluorolog 3-11 spectrofluorometer equipped with a 450 W xenon lamp. Excitation and emission were measured at room temperature directly on the colloidal suspensions, after dialysis.

Excitation spectra were recorded between 350 and 600 nm, monitoring the 5 D 0 f 7 F 2 emission of Eu 3+ at λ em ) 612 nm (spectral bandwidth ) 2 nm). Emission spectra were recorded in the 500-700 nm range, with a spectral bandwidth of 1 nm, under selected excitation in the 7 F 0 f 5 L 6 transition of Eu 3+ at λ ex ) 392.8 nm or in the 7 F 0 f 5 D 2 transition at λ ex ) 464.2 nm.

The transient characteristics of the emitting level 5 D 0 of Eu 3+ were investigated with the phosphorimeter FL-1040, equipped with a UV xenon flash tube. Emission decays were analyzed at chosen λ ex and λ em on a time interval of 3.5 ms. The time resolution imposed by the apparatus in the experimental conditions employed is 30 µs.

Results and Discussion

3.1. Physicochemical Characterization. Several apatite suspensions were prepared in the presence of AEP with increasing europium contents corresponding to initial Eu/ (Eu + Ca) molar ratios of 0, 1, 1.5, 2, 2.5, 4, 7, 10, and 100%. The suspensions prepared from initial ratios lower than 2.5% were translucent colloids, highly stable over time (greater than 6 months). In contrast, beyond this initial europium content in the solution, a noticeable increase of the suspension viscosity was observed, leading to a rapid sedimentation in the case of 100%.

The XRD patterns obtained on the purified freeze-dried suspensions are reported in Figure 1. The patterns corresponding to molar ratios in the initial reaction mixture up to 2.5% clearly exhibit diffraction peaks characteristic of an apatite structure, and no additional crystallized phase could be detected. Ad-ditionally, the crystallinity state of these samples was found to decrease with increasing Eu contents in the initial mixture, which is, for example, visible in the 2θ ranges of 35-41°and 53-63°. This point probably unveils some inhibitory effect of europium on apatite crystal growth, as already evidenced for other ions. 49 A similar inhibitory effect of AEP molecules can also be seen by comparing the 0% Eu colloidal suspension to its noncolloidal AEP-free counterpart (see Figure 1), which is in agreement with a previous report for AEP-containing apatite colloids. 45 On the contrary, for initial Eu/(Eu + Ca) molar ratios greater than 2.5%, a drastic modification of the XRD pattern was seen, and apatite could no more be detected in these systems, which were found to approach the structure of europium phosphate (comparison to JCPDS card No. 20-1044 relative to EuPO 4 • H 2 O), despite a poor crystallinity state. Chemical analyses performed on the sample corresponding to 100% Eu revealed a Eu/PO 4 molar ratio close to unity (experimental value ) 0.96), therefore, corroborating the hypothesis of europium phosphate formation in this case.

FTIR spectra were then recorded on the purified freeze-dried samples corresponding to the varying europium doping rates (Figure 2). For initial Eu/(Eu + Ca) molar contents between 0 and 2%, the characteristic absorption bands of apatite were observed, especially around 1095, 1033, 601, and 563 cm -1 , assigned to the PO 4 3-ν 3 and ν 4 vibration modes. The peak observed around 875 cm -1 can be assigned to HPO 4 2-ions. The broad bands observed are, however, only moderately welldefined, with a decreasing resolution as the europium doping rate increases, which can be related to a rather low crystallinity state, in accordance with the XRD findings. The apatitic nature of the phase in presence was also checked by Raman spectroscopy as the characteristic PO 4 3-ν 1 band was observed at 961 cm -1 .

In contrast, the FTIR spectra obtained for Eu/(Eu + Ca) ratios greater than 4% exhibit sharp alterations, revealing strong modifications for elevated europium contents. In particular, the spectrum recorded for 100% Eu, with pronounced bands at 538, 628, and 1082 cm -1 , was found to be characteristic of hydrated europium phosphate; this was checked by comparison with a reference EuPO 4 .H 2 O compound prepared by precipitation at room temperature from europium chloride and ammonium dihydrogen phosphate. The sample prepared with a Eu/ (Eu + Ca) ratio of 2.5% was found to be in an intermediary situation, with apatite obviously present beside europium phosphate. This observation indicates that a finer analysis can be drawn from FTIR data as compared to XRD results where the presence of europium phosphate could hardly be detected for the sample corresponding to the initial Eu/(Eu + Ca) ratio of 2.5% Eu.

The examination of the FTIR spectra for the single-phased systems corresponding to initial Eu/(Eu + Ca) ratios ranging from 0 to 2% also showed the presence of additional peaks, in particular, at 754 cm -1 . These bands are not assignable to apatite nor to europium phosphate. These additional features can be unambiguously linked to the presence of AEP molecules in the colloids, in the form of Ca(AEP) 2 , as was shown earlier. 45 These findings thus unveil the existence of Ca 2+ (AEP -) 2 complexes on the surface of the apatite nanocrystals, as was previously shown for europium-free apatite colloids. 45 Chemical analyses were carried out on the purified samples corresponding to Eu/(Eu + Ca) starting ratios from 0 to 2%. The final Eu/(Eu + Ca) ratios in the colloids were then found in the range of 0-2.9 atomic %. The Ca, Eu, and mineral phosphate contents were quantified, enabling us to evaluate the overall (Ca + Eu)/P ratio of the colloids. This ratio was found in all cases between 1.35 and 1.50. These values are noticeably lower than the value of 1.67 corresponding to stoichiometric hydroxyapatite; they, therefore, point out the nonstoichiometric nature of the apatite phase, thus confirming its biomimetic nature.

The possibility to incorporate europium in the apatite structure has already been mentioned by several authors, 42,44,45 although generally for apatite compounds prepared at higher temperature. Several substitution mechanisms can be considered 43 for explaining the incorporation of Eu 3+ ions in Ca 2+ sites. In the absence of monovalent or tetravalent ions in the medium, four distinct mechanisms can be distinguished: the replacement of three Ca 2+ by two Eu 3+ (mechanism no.1), the deprotonation of some HPO 4 2-ions into PO 4 3-(mechanism no. 2), and the additional incorporation of OH -(mechanism no. 3) or of oxygen O 2-ions (mechanism no. 4). The existence of oxygen ions O 2- in Eu-doped apatites, as involved in mechanism no. 4, has indeed been reported and should thus not be a priori discarded. [42][43][44] It should be noted that a combination of two or more of these mechanisms may also be involved. To investigate some more the Eu incorporation mechanism, a series of noncolloidal apatite suspensions were prepared in similar conditions, but in the absence of AEP in the medium. Again, increasing Eu contents in the range of 0-2% were considered. After chemical analyses were performed on several specimens of each sample, the (Ca + Eu)/P molar ratio of the apatite phase was evaluated for these noncolloidal samples. The results obtained pointed out a slight decrease of this ratio upon Eu incorporation: typically from 1.66 down to 1.57 for Eu starting doping rates increasing from 0 to 2%. Based on the knowledge of Eu/(Ca + Eu) and (Ca + Eu)/P ratios for the 0, 1, 1.5, and 2% Eu samples, one can thus estimate their chemical composition by considering consecutively each of the four possible incorporation mechanisms. Interestingly, these determinations indicated that all four mechanisms remained possible for explaining the incorporation of Eu 3+ ions in Ca 2+ sites. This is, for instance, exemplified in Figure 3 in the case of the sample corresponding to a starting doping rate of 1.5% Eu. A preferential mechanistic scheme thus cannot be derived at this point. It should also be noted that the preferential mechanistic scheme for Eu incorporation may depend, to some extent, on the doping rate. The observation of a maximum limit of Eu incorporation does not seem to be directly explainable based only on mechanistic considerations: this limit probably originates from other factors, such as thermodynamically driven destabilization of the system, possibly linked to differences between Eu-O and Ca-O bond lengths. Additional work specifically dedicated to examine these aspects will be needed to investigate this point, which lies beyond the scope of the present paper. Although the presence of AEP in the colloidal synthesis route leads to less-crystallized apatite phases, linked to some inhibitory effect of AEP pointed out from XRD analyses, it seems reasonable to expect a similar situation in terms of Ca/Eu substitution.

The amount of AEP contained in the colloids was determined from the amount of nitrogen titrated by way of elemental microanalyses. The AEP content was found to represent 8-11 wt % of the colloidal particles, leading to an AEP/apatite molar ratio in the range of 0.7-0.8. It is interesting to note that this ratio is lower than the one observed by Bouladjine et al. 45 for europium-free apatite colloids prepared at 80 °C. These findings can probably be explained by the differences existing in the synthesis protocols used in the two studies, especially in terms of starting ionic concentrations and aging temperatures. The location of the AEP molecules on the surface of the apatite crystals was evidenced by Bouladjine et al. 45 who showed the positive surface charge of the colloids obtained in the presence of AEP and the decrease in size upon increasing the AEP content. Interestingly, the zeta potential of the colloids prepared in the present work was also found to be clearly positive (e.g., +12.4 mV for the sample corresponding to the initial doping of 1.5% Eu), thus showing again the exposure of the ammonium groups -NH 3 + from AEP molecules toward the solution. Also, a decrease of the initial AEP concentration provoked a sedimentation phenomenon for AEP/(Ca + Eu) ratios lower than 0.2, whereas stable colloidal suspensions were obtained beyond 0.4. This sedimentation can then most probably be explained by an insufficient electrosteric repulsion between adjacent particles when the surface content in AEP molecules decreases beyond a limit value.

Transmission electron microscopy (TEM) observations were performed on the dialyzed colloids in order to inspect the constitutive nanocrystals within the colloidal particles. In a first step, the study was conducted on colloidal suspensions with Eu contents ranging from 0 to 1.5% (prepared with an initial ratio AEP/(Ca + Eu) ) 1) to study the effect of the introduction of Eu 3+ ions into the mineral host matrix. TEM observations showed no obvious differences when varying the europium content (see Figure 4) for samples doped with 0 and 1.5% Eu. In all cases, the micrographs recorded at low magnification revealed very homogeneous crystal aspects with an ellipsoidal morphology and with dimensions around 28 nm in length and 9 nm in width. These observations attest that the introduction of europium in this Eu/(Ca + Eu) range has no obvious impact on the size and morphology of the individual nanocrystals constituting the colloidal particles.

In a second step, the effect of a change of the initial AEP/ (Ca + Eu) ratio in the range of 0.4-1 was followed by TEM in order to inspect the possible influence of the amount of AEP on the size and aspect of individual nanocrystals. The results obtained showed (Figure 5) very similar features for AEP/ (Ca + Eu) ) 0.4, 0.6, and 1, indicating that the presence of AEP in these conditions did not affect noticeably the size and aspect of the individual apatite nanocrystals.

Numerous studies have, however, shown the general tendency of apatite nanocrystals to agglomerate. Although TEM observations showed no impact of the presence of AEP molecules on the size of the constitutive nanocrystals, differences in the agglomeration state may occur, as was already observed for europium-free samples. 45 To determine the size of the colloidal particles obtained and to follow, thereby, the agglomeration state of apatite, granulometry measurements were carried out on the colloids by dynamic light scattering (DLS), leading to hydrodynamic radii, D h .

The results (Figure 6) showed that, for initial AEP/(Ca + Eu) ratios between 1 and 0.6, the hydrodynamic diameter (D h ) was close to 30 nm. This value is very close to the individual crystal size estimated from TEM, indicating that, for these AEP contents, the crystal agglomeration is extremely limited. These findings can most probably be explained by the strong electrosteric repulsive effect induced by the presence of an important amount of AEP on the surface of the apatite nanocrystals. In contrast, for decreasing AEP/(Ca + Eu) ratios from 0.6 to 0.2, the value of D h increased rapidly up to about 95 nm. Such sizes, greater than the ones measured for individual crystals by TEM, unveil an increased agglomeration state of the apatite nanocrystals when the amount of AEP decreases: the presence of these molecules controls the agglomeration state of the colloidal particles. In other words, the particle size for such apatite colloids may be tailored by varying the synthesis conditions.

3.2. Luminescence Properties. The luminescence properties of the colloids prepared in this work were then investigated.

Excitation spectra were first recorded by monitoring the red luminescence intensity at 612 nm (main emission line). The maximum emission was found for an excitation performed at 392.8 nm, a wavelength close to the visible light domain. This wavelength corresponds to the direct excitation of Eu 3+ ions from the ground state 7 F 0 to the 5 L 6 excited level: it is usually the most intense of the intraconfiguration absorption lines of Eu 3+ (4f 6 configuration).

As expected, a secondary excitation peak, although less intense, is also observed at 464.2 nm. This peak is assignable to the 7 F 0 f 5 D 2 absorption and lies within the visible domain. These observations show the possibility to use such luminescent nanoprobes for studying biological tissues because they only require low-energy excitation, preventing the deterioration of live cells.

Luminescence emission spectra were then recorded for suspensions corresponding to the starting Eu/(Eu + Ca) ratios of 1, 1.5, and 2%, under excitation at 392.8 nm (Figure 7). The observed spectral features are easily ascribed to the 4f-4f transition within the 4f 6 configuration of Eu 3+ . In all cases, three domains can be distinguished: 575-580 nm, 583-603 nm, and 605-627 nm, corresponding, respectively, to the transitions 5 D 0 f 7 F 0 , 5 D 0 f 7 F 1 , and 5 D 0 f 7 F 2 characteristic of the Eu 3+ ion. Also, the global emission intensity was found to increase noticeably with the level of europium doping.

The 5 D 0 f 7 F 0 transition located at 578 nm has been studied, in particular, because the energy levels 5 D 0 and 7 F 0 are both nondegenerated, meaning that only a single peak for the 5 D 0 f 7 F 0 transition should appear if all the Eu 3+ ions were in the same crystal field environment. Besides, the maximum numbers of emission lines are three and five per crystal field environment for 5 D 0 f 7 F 1 and 5 D 0 f 7 F 2 , respectively. For all three cases, only one component is recorded for 5 D 0 f 7 F 0 , then three distinct components for 5 D 0 f 7 F 1 , and four or five for 5 D 0 f 7 F 2 , with a considerable overlapping of the lines, due to inhomogeneous broadening as it is usual in poorly crystallized samples.

It is, however, worth mentioning that, for the suspension obtained from an initial Eu/(Eu + Ca) ratio of 2%, a significant change was observed for each transition as compared with the other two suspensions. The intensity of luminescence was found to notably increase, and as a general observation, the lines are narrower than for the two other samples. Moreover, the overall shape of 5 D 0 f 7 F 2 is modified with relatively more intensity at 610 and at 624 nm. These findings indicate that the immediate chemical environment of europium in this colloid is partly different from the environment found in the other two suspensions. To shed some light on this aspect, luminescence emission spectra of EuPO 4 • H 2 O and Eu(OH) 3 reference compounds were recorded and compared with the emission spectrum of the 2% Eu suspension (Figure 8). The spectra being poorly resolved, it may not be concluded if one or the other of the two phases appears exclusively in the 2% colloid: more probably, a mixture of both is present, beside the apatite, in this sample.

Such observations show that luminescence spectroscopy proves to be an interesting complementary tool for sample characterization, exploiting europium as an internal probe. Indeed, the presence of traces of such secondary phases had only been suggested from FTIR analyses beyond a starting Eu/ (Ca + Eu) ratio of 2.5%.

The luminescence lifetime of the europium 5 D 0 level measured on the same set of samples was also studied, and Figure 9 displays the decays observed for suspensions corresponding to an initial Eu/(Eu + Ca) ratio of 1, 1.5, and 2%. As observed on the spectra, it may be noticed that the decays for 1% and 1.5% Eu are essentially the same, whereas for 2% Eu, the emission decays more slowly. In the figure (Figure 9), the I(t) curves have been displayed on a logarithmic scale, showing that they are not purely monoexponential. We have, nevertheless, measured the average decay time τ as the time for which I(τ) ) I 0 /exp(1), leading to 0.73 ( 0.03 ms for 1% or 1.5% Eu and 1.00 ( 0.05 ms for 2% Eu. This observation may be linked to the above results, suggesting the presence of secondary phases beside apatite for this sample, leading to modified luminescence properties.

It is interesting to note that, in all cases, the luminescence exponential decay was found to be of the order to the millisecond. This order of magnitude is customary, in the case of lanthanide-doped systems, due to the intrinsic properties of Ln 3+ luminescent centers, and it enables one to envision applications as luminescent nanoprobes for biological applica-tions because the autofluorescence of the biological medium exhibits a luminescence decay time of the order of nanoseconds. In a second step, the luminescence properties, both in terms of excitation/emission and lifetime, of colloidal suspensions obtained at constant europium content (initial doping ) 1.5% Eu), but from varying initial amounts of AEP in the medium, were followed. AEP/(Ca + Eu) molar ratios of 0.4, 0.6, 0.8, and 1 were tested. The results obtained indicated that no obvious differences could be noticed between the three suspensions, suggesting that the chemical environment of europium was essentially unchanged.

Conclusions

Europium-doped calcium phosphate apatite-based colloids were produced in the presence of a phospholipid moiety, 2-aminoethylphosphoric acid (AEP), with varying europium doping rates, and their physicochemical characteristics were investigated in detail. The experimental data obtained for suspensions prepared from an initial Eu/(Ca + Eu) molar ratio up to 2% converge to indicate that these colloids consist of biomimetic apatite nanocrystals covered with AEP molecules. Moreover, the agglomeration state of apatite crystals was found to be closely dependent on the amount of AEP in the system, and the mean particle size can be tailored between 30 and 100 nm. The colloidal suspensions as prepared were found to be stable over time beyond at least 6 months.

The luminescence properties of these nanoprobes were analyzed in terms of excitation, emission, and lifetime. The typical luminescence features of europium-doped systems were witnessed, with narrow emission peaks and long luminescence lifetimes. Our observations support the location of europium in a common crystallographic environment for all the colloids prepared.

The potential of such systems in the field of optical imaging related to biology will then have to be investigated. Supporting Information Available: Emission spectra of apatite colloids doped with Eu/(Ca + Eu) initial molar ratios of 1, 1.5, and 2% Eu under excitation at 392.8 nm; luminescence decay profiles of the 5 D 0 (Eu 3+ ) level for three apatite col-loidss1, 1.5, and 2% Eusunder excitation at 392.8 nm and at ambient temperature; and TEM micrograph for apatite colloids prepared from Eu/(Ca + Eu) ) 1.5%. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 1 .

 1 Figure 1. XRD patterns for apatite colloids prepared with increasing Eu/(Ca + Eu) initial molar ratios and for a noncolloidal reference (AEPfree).

Figure 2 .

 2 Figure 2. FTIR spectra for apatite colloids prepared with increasing Eu/ (Ca + Eu) initial molar ratios and for a noncolloidal reference (AEP-free).

Figure 3 .

 3 Figure 3. Chemical composition for the 1.5% Eu sample (starting doping rate) evaluated from the four different incorporation mechanisms.

Figure 4 .

 4 Figure 4. TEM observations (scale bar ) 500 nm) of the apatite colloids with Eu/(Ca + Eu) initial ratios of 0% (a, b) and 1.5% (c, d), prepared from AEP/(Ca + Eu) ) 1.

Figure 5 .

 5 Figure 5. TEM observations (scale bar ) 200 nm) of apatite colloids doped with Eu/(Ca + Eu) initial molar ratios at 1.5% and AEP/ (Ca + Eu) ) 0.8 (a), 0.6 (b), and 0.4 (c).

Figure 6 .

 6 Figure 6. Effect of the AEP/(Ca + Eu) initial molar ratio on the hydrodynamic diameter of colloidal particles.

Figure 7 .

 7 Figure 7. Emission spectra of apatite colloids doped with Eu/(Ca + Eu) initial molar ratios of 1% (a), 1.5% (b), and 2% (c) of europium Eu 3+ under excitation at 392.8 nm.

Figure 8 .

 8 Figure 8. Emission spectra of a Eu 3+ -doped apatite colloid with a Eu/(Ca + Eu) initial molar ratio of 2% (a) and for reference compounds EuPO 4 • H 2 O (b) and Eu(OH) 3 (c), under excitation at 392.8 nm.

Figure 9 .

 9 Figure 9. Luminescence decay profiles of the 5 D 0 (Eu 3+ ) level for three apatite colloids: 1% (a), 1.5% (b), and 2% (c), under excitation at 392.8 nm and at ambient temperature.


Paul Gabarre

Christopher Loens

Yanis Tamzali

Frédéric Benoit Barrou

Gabarre Paul

Loens Christopher

Tamzali Yanis

Barrou Benoit

Jaisser Frédéric

Tourret Jérôme
email: jerome.tourret@aphp.fr

Immunosuppressive therapy after solid organ transplantation and the gut microbiota: bidirectional interactions with clinical consequences

Keywords: Gut microbiota GUS: B-glucuronidase IST: Immunosuppressive treatment KT : Kidney transplantation LPS : Lipopolysaccharide MLC2: Myosin light chain 2 MLCK: Myosin light chain kinase Tumor necrosis factor UTI: Urinary tract infection immunosuppressive drugs, gut microbiota, side effect, bacteria, solid organ transplantation

published or not. The documents may come

Immunosuppressive therapy after solid organ transplantation and the gut microbiota: bidirectional interactions with clinical consequences

1.

The gut microbiota in solid organ transplantation: why should we care?

The gut microbiota (GM) is the total microbial mass that colonizes the digestive tract of a host. It is composed of trillions of bacteria, viruses, archaea, fungi that are distributed throughout the digestive tract with different abundances [START_REF] Thursby | Introduction to the human gut microbiota[END_REF][START_REF] Savage | Microbial Ecology of the Gastrointestinal Tract[END_REF] . Our knowledge of the GM has dramatically increased over the last decades principally because of the development of new genetic tools [START_REF] Lynch | The Human Intestinal Microbiome in Health and Disease[END_REF] .

Solid organ transplantation can only be considered medically when accompanied by the longterm use of immunosuppressive treatments (IST), to avoid graft rejection. However, ISTs are responsible for major side effects such as infections, diabetes and high blood pressure.

Yet, transplantation and ISTs prescribed after transplantation have been shown to be associated with changes in the GM. The GM has a wide array of functions that benefit the host, such as digestion and energy harvesting [START_REF] Turnbaugh | An obesity-associated gut microbiome with increased capacity for energy harvest[END_REF] , protection from colonization by pathogens [START_REF] Kamada | Control of Pathogens and Pathobionts by the Gut Microbiota[END_REF] , training of the immune system [START_REF] Hill | Intestinal Bacteria and the Regulation of Immune Cell Homeostasis[END_REF] , fat storage [START_REF] Backhed | The gut microbiota as an environmental factor that regulates fat storage[END_REF] , neuropsychological development [START_REF] Fung | Interactions between the microbiota, immune and nervous systems in health and disease[END_REF] , homeostasis [START_REF] Heijtz | Normal gut microbiota modulates brain development and behavior[END_REF] and the metabolism of xenobiotics [START_REF] Claus | Colonization-Induced Host-Gut Microbial Metabolic Interaction[END_REF] . The GM is capable of resilience, i.e. it has the ability to restore a homeostatic balance between the different sub-populations despite external perturbations [START_REF] Vanlancker | 5-Fluorouracil and irinotecan (SN-38) have limited impact on colon microbial functionality and composition in vitro[END_REF] . Durable modifications of the GM, called dysbiosis, have been associated with several chronic diseases, such as cardiovascular (high blood pressure [START_REF] Li | Gut microbiota dysbiosis contributes to the development of hypertension[END_REF][START_REF] Oyama | Gut microbiota and hypertension[END_REF][START_REF] Yang | Gut Dysbiosis Is Linked to Hypertension[END_REF][START_REF] Honour | Historical perspective: gut dysbiosis and hypertension[END_REF] ), metabolic (diabetes mellitus [START_REF] Consortium | Aberrant intestinal microbiota in individuals with prediabetes[END_REF][START_REF] Tilg | Microbiota and diabetes: an evolving relationship[END_REF][START_REF] Larsen | Gut Microbiota in Human Adults with Type 2 Diabetes Differs from Non-Diabetic Adults[END_REF][START_REF] Cani | Metabolic Endotoxemia Initiates Obesity and Insulin Resistance[END_REF] and obesity [START_REF] Vanlancker | 5-Fluorouracil and irinotecan (SN-38) have limited impact on colon microbial functionality and composition in vitro[END_REF] ) and neurological disorders [START_REF] Parashar | Gut microbiota: Implications in Parkinson's disease[END_REF][START_REF] Hu | Alzheimer's disease and gut microbiota[END_REF] , and inflammatory bowel diseases [START_REF] Sokol | Specificities of the fecal microbiota in inflammatory bowel disease[END_REF][START_REF] Mazmanian | A microbial symbiosis factor prevents intestinal inflammatory disease[END_REF][START_REF] Barnich | Role of bacteria in the etiopathogenesis of inflammatory bowel disease[END_REF] .

It has been demonstrated that some drugs used in human health interact with the GM. On the one hand, treatments with antibiotics [START_REF] Maurice | Xenobiotics shape the physiology and gene expression of the active human gut microbiome[END_REF][START_REF] Modi | Antibiotics and the gut microbiota[END_REF][START_REF] Pérez-Cobas | Gut microbiota disturbance during antibiotic therapy: a multi-omic approach[END_REF] , chemotherapies [START_REF] Zwielehner | Changes in Human Fecal Microbiota Due to Chemotherapy Analyzed by TaqMan-PCR, 454 Sequencing and PCR-DGGE Fingerprinting[END_REF][START_REF] Montassier | Chemotherapy-driven dysbiosis in the intestinal microbiome[END_REF][START_REF] Montassier | 16S rRNA Gene Pyrosequencing Reveals Shift in Patient Faecal Microbiota During High-Dose Chemotherapy as Conditioning Regimen for Bone Marrow Transplantation[END_REF][START_REF] Bastard | Fecal microbiota transplantation reverses antibiotic and chemotherapyinduced gut dysbiosis in mice[END_REF] and many others modify the microbiota, which can lead to dysbiosis and adverse events such as obesity [START_REF] Million | Lactobacillus reuteri and Escherichia coli in the human gut microbiota may predict weight gain associated with vancomycin treatment[END_REF] . On the other hand, the anti-diabetes drug metformine [START_REF] Wu | Metformin alters the gut microbiome of individuals with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug[END_REF] and checkpoint blocker ipilimumab [START_REF] Vetizou | Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota[END_REF] exert their beneficial effects by modifying the GM, which in turns affects the host physiology.

According to the Global Observatory on Donation and Transplantation, more than 130,000 solid organ transplantations have been performed in 2016, including nearly 90,000 (69%) kidney transplantations [START_REF]The global observatory on donation and transplantation[END_REF] , exposing recipients to prolonged ISTs to avoid graft rejection [START_REF] Zaza | Personalization of the Immunosuppressive Treatment in Renal Transplant Recipients: The Great Challenge in "Omics[END_REF] .

Bidirectional interactions between the gut microbiota and the host immunology

The GM interacts with host immunity through a complex crosstalk. The involvement of the GM in the maturation of the immune system was demonstrated by the study of axenic (i.e.

germ-free) mice, which develop an immature immune system with atrophic lymphatic nodes, spleen and Peyer's patches [START_REF] Round | The gut microbiota shapes intestinal immune responses during health and disease[END_REF] .

Innate immunity is generally affected as well. Microbial peptidoglycans and flagellins induce the production of anti-microbial peptides by intestinal epithelial cells in the lumen of the digestive tract. The buildup of this immunity involves the transcription factor NF-κB and the Toll-like Receptor 5 of dendritic cells, respectively [START_REF] Levy | Dysbiosis and the immune system[END_REF] . The GM also impacts adaptative immunity, such as the inflammation/tolerance balance (Th1-2-17/Tregs-Th1) [START_REF] Sommer | The gut microbiota--masters of host development and physiology[END_REF] . As an example, Bacteroides fragilis -one of the anti-inflammatory members of the GM -induces Treg lymphocytes and attenuates colitis [START_REF] Knauf | Immunity, microbiota and kidney disease[END_REF] . The polysaccharide A at the surface of Bacteroides fragilis is recognized by the TLR2 receptor of CD4 + cells [START_REF] Round | The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota[END_REF] , resulting in their differentiation into Treg lymphocytes. Tregs repress the pro-inflammatory activity of Th17 cells through the local and systemic secretion of interleukin 10 41 .

This GM-driven redirection of the inflammation/tolerance balance has clinical consequences.

For example, a pro-inflammatory GM may be involved in the progression of chronic kidney disease [START_REF] Anders | The intestinal microbiota, a leaky gut, and abnormal immunity in kidney disease[END_REF][START_REF] Vaziri | Chronic kidney disease alters intestinal microbial flora[END_REF] ; in a 'vicious circle', uremic toxins modify the microbial composition of the gut, which promotes systemic inflammation and the further progression of kidney lesions.

Conversely, the host immune system controls the GM; when immunosuppressed mice are colonized with opportunistic pathogens [START_REF] Brandl | Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits[END_REF] , they develop septicemia with digestive commensals [START_REF] Slack | Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism[END_REF] .

The importance of the bidirectional interactions between the GM and the host's immunity needs to be explored in the specific context of organ transplantation and IST. If the host's immune system controls the GM, what is the impact on the GM of drugs which interfere with the immune system?

Modifications of the GM after liver transplantation

Zhong When-Wu et al. studied the fecal microbiota of liver transplant recipients, as compared to healthy volunteers and cirrhotic patients [START_REF] Wu | Changes of gut bacteria and immune parameters in liver transplant recipients[END_REF] . They highlighted that liver transplantation was associated with gut dysbiosis characterized by a decrease of the total bacterial mass, and of beneficial bacteria such as Bifidobacterium spp., Faecalibacterium prausnitzii and Lactobacillus spp. and an increase of Enterobacteriaceae and Enterococcus spp. However, 13-24 months after liver transplantation, all bacteria populations tended to return to a normal level, except for Enterococcus spp. which was responsible for infections (urinary tract, surgical site and bacteremia) after the liver transplantation 47 .

Modifications of the GM after kidney transplantation

Description of the GM alterations after kidney transplantation

Fricke et al. [START_REF] Fricke | Human Microbiota Characterization in the Course of Renal Transplantation: Microbiota Changes During Renal Transplantation[END_REF] studied the evolution of the gut, urine and oral microbiotas after kidney transplantation (KT) in 60 patients. They observed an early switch in the GM composition after KT. Modifications were more extensive between before KT and one month after KT than between 1 month and 6-month after KT, and persisted over time, suggesting a loss of the GM resilience.

In another study, Bacteroidetes relative abundance appeared to be reduced in KT recipients, while Proteobacteria were increased [START_REF] Lee | Gut Microbial Community Structure and Complications Following Kidney Transplantation: A Pilot Study[END_REF] . The Proteobacteria phylum contains opportunistic pathogens such as Salmonellae, and Escherichia coli, and is considered a signature of an unstable and pro-inflammatory GM [START_REF] Shin | Proteobacteria: microbial signature of dysbiosis in gut microbiota[END_REF] .

Adverse events after kidney transplant correlate with gut microbiota modifications

Pre-transplant gut microbiota is associated with post-transplant clinical events

In the study of Fricke et al. [START_REF] Fricke | Human Microbiota Characterization in the Course of Renal Transplantation: Microbiota Changes During Renal Transplantation[END_REF] , those transplant patients who experienced rejection had, prior to transplantation, a significant scarcity of four bacterial genera from the Firmicutes phylum (Anaerotruncus, Coprobacillus, Coprococcus and an unknown member of the Peptostreptococcaceae), compared to patients without rejection events. Anaerotruncus was also significantly depleted prior to KT in the GM of patients who experienced posttransplantation infection.

Finally, in our recent study, patients who developed New Onset Diabetes After (kidney)

Transplantation tended to have a higher carriage of fecal Lactobacillus sp. prior to KT, and also had a lower abundance of F. prausnitzii than patients who did not develop diabetes [START_REF] Lecronier | Gut microbiota composition alterations are associated with the onset of diabetes in kidney transplant recipients[END_REF] .

Post-transplant gut microbiota differences are associated with post-transplant clinical events

Lee et al. Interestingly, some of these bacteria are producers of short chain fatty acids, which are considered beneficial for the host [START_REF] Koh | From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites[END_REF] .

It is important to note that the causality of these associations has not been investigated. Yet, these studies suggest that the GM is modified after solid organ transplantation (SOT) and IST and raise the question of the implication of both pre-and post-transplant microbiota in the onset of the main post-transplant complications. Therefore, a better understanding of GM modifications after SOT/IST appears to be required.

SOT is associated with many factors possibly involved in the modification of the GM, such as anti-infectious prophylaxis, general anesthesia, dietary changes, and the restoration of the transplanted organ function (Figure 1). Recently, several studies have demonstrated that IST also modified the GM, and this review focuses on this aspect.

Corticosteroids

Glucocorticosteroids (GCs) remain a major part of anti-rejection treatment strategies following a SOT 54 .

Gut microbiota changes induced by corticoid treatment in rodents

In a mouse model, glucocorticoids decreased bacterial richness and diversity (Chao1 and Shannon indexes) [START_REF] Wu | Chronic glucocorticoid treatment induced circadian clock disorder leads to lipid metabolism and gut microbiota alterations in rats[END_REF] and altered the global composition of the GM [START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF] .

At the phylum level, the literature is heterogeneous. Our study showed an increased Firmicutes/Bacteroidetes ratio 56 , while other studies found a decrease of Firmicutes 55 , Bacteroidetes, Actinobacteria, alpha and gamma Proteobacteria 55 and Deferribacteres 57 .

At deeper levels, GCs were reported to increase the relative abundance of fecal Clostridiales, Lactobacillus [START_REF] Wu | Chronic glucocorticoid treatment induced circadian clock disorder leads to lipid metabolism and gut microbiota alterations in rats[END_REF] , Anaerostipes [START_REF] He | Alterations of the Gut Microbiota Associated With Promoting Efficacy of Prednisone by Bromofuranone in MRL/lpr Mice[END_REF] , Bifidobacterium [START_REF] Huang | Using corticosteroids to reshape the gut microbiome: implications for inflammatory bowel diseases[END_REF] and decreased Oscillospira, Bilophila, and Rikenella 57 . In addition, two studies found a decrease of Mucispillirum [START_REF] Steiner | Steroids in kidney transplant patients[END_REF][START_REF] Wu | Chronic glucocorticoid treatment induced circadian clock disorder leads to lipid metabolism and gut microbiota alterations in rats[END_REF] , mucin degrading bacteria [START_REF] Rodríguez-Piñeiro | The colonic mucus protection depends on the microbiota[END_REF] that are involved in the maturation and the activation of T cells through an interaction with antigen presenting cells [START_REF] Bunker | Innate and adaptive humoral responses coat distinct commensal bacteria with immunoglobulin A[END_REF] .

The GM is physiologically capable of resilience, defined as its ability to return to an initial state after a disturbance [START_REF] Sommer | The resilience of the intestinal microbiota influences health and disease[END_REF] . Kim et al. demonstrated that dexamethasone (DXM) administration in mice increased the delay before GM demonstrated resilience, exposing mice to more severe Clostridium difficile infection [START_REF] Kim | A Detrimental Role of Immunosuppressive Drug, Dexamethasone, During Clostridium difficile Infection in Association with a Gastrointestinal Microbial Shift[END_REF] . Finally, GCs decreases Clostridium sensu stricto abundance in the ileum [START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF] .

Disruption of circadian rhythms as a potential mechanism of GC-induced dysbiosis

Many organisms undergo 24 hours cycles, with markers varying in a circadian rhythm [START_REF] Edgar | Peroxiredoxins are conserved markers of circadian rhythms[END_REF] .

Similarly, the GM exhibits daily fluctuations and any perturbations of the circadian rhythm that the host experiences (such as jetlag) is responsible for a loss of rhythmicity associated with dysbiosis [START_REF] Thaiss | Transkingdom Control of Microbiota Diurnal Oscillations Promotes Metabolic Homeostasis[END_REF] . Interestingly, endogenous glucocorticoids regulate the circadian rhythmicity [START_REF] So | Glucocorticoid regulation of the circadian clock modulates glucose homeostasis[END_REF] and exogenous GCs such as dexamethasone are strong perturbators of circadian rhythm-related genes, thus counteracting endogenous steroids 66 . Wu et al. [START_REF] Wu | Chronic glucocorticoid treatment induced circadian clock disorder leads to lipid metabolism and gut microbiota alterations in rats[END_REF] studied the effect of chronic administration of GCs on the circadian rhythm and the GM. After seven weeks of treatment, rats exhibited the following changes: alteration of the global composition of the GM characterized by a decreased richness and diversity, a decrease of the main phyla as described in the preceding section, attenuation of the weight gain, an accumulation of fat and loss of their circadian rhythm.

Glucocorticoids alter the intestinal barrier

The intestinal barrier is composed of three layers: the lamina propria, epithelial intestinal cells, and the mucus layer. The mucus layer plays a major role in pathogen resistance [START_REF] Desai | A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility[END_REF] , by the expression of virulence genes of pathogens and in the nutrition of the GM [START_REF] Sicard | Interactions of Intestinal Bacteria with Components of the Intestinal Mucus[END_REF] . The intestinal barrier maintains a physiological distance between the GM and the body by chemical, mechanical and immunological means and also participates in the regulation of the GM [START_REF] Ghim | Mechanisms regulating intestinal barrier integrity and its pathological implications[END_REF][START_REF] Schroeder | Fight them or feed them: how the intestinal mucus layer manages the gut microbiota[END_REF] .

Treatment with DXM is responsible for a decreased expression of Muc2, the main component of the colonic mucus [START_REF] Wu | Chronic glucocorticoid treatment induced circadian clock disorder leads to lipid metabolism and gut microbiota alterations in rats[END_REF][START_REF] Huang | Using corticosteroids to reshape the gut microbiome: implications for inflammatory bowel diseases[END_REF] . Interestingly, Muc2 expression was not altered when DXM was given to germ-free mice, suggesting that downregulation of Muc2 by DXM implicates the GM. Conversely, fecal microbiota transplantation from DXM-treated mice led to an increased expression of Muc2 and a reduced inflammation in recipient mice that were genetically susceptible to colitis (IL-10 -/-knockouts).

GCs also alters the innate immunity of the intestinal barrier. Indeed, prednisolone was responsible for a decreased expression of C-type Lectins RegIIIβ and RegIIIɤ 56 , which are involved in the control of intestinal bacterial proliferation and the host's immune response to the GM [START_REF] Muniz | Intestinal antimicrobial peptides during homeostasis, infection, and disease[END_REF] . Moreover, administration of DXM to rats decreased the level of biliary IgA 72 and decreased IgA bacterial coating [START_REF] Alverdy | The effect of glucocorticoid administration on bacterial translocation. Evidence for an acquired mucosal immunodeficient state[END_REF] , a process that neutralizes pathogenic bacteria 74 . Finally, an in vitro study showed that GCs prevented a TNF-α-induced increase in intestinal epithelial permeability. The binding of GCs on their receptor inhibited TNF-α-induced expression of

Myosin Light Chain Kinase (MLCK). MLCK phosphorylates and activates Myosin Light

Chain 2 (MLC2), which is responsible for the contraction of the peri-junctional actin-myosin filaments, leading to the dysfunction of the tight junctions. As a consequence, GCs inhibited TNFα-induced tight junction dysfunction 75 (see Figure 2 and Table 1 for an overview of the effects of the various ISTs on the GM discussed in this Review).

The gut microbiota is involved in GC metabolism and efficacy

In the gut, endogenous GCs are metabolized by Clostridium scindens [START_REF] Ridlon | Clostridium scindens : a human gut microbe with a high potential to convert glucocorticoids into androgens[END_REF] . In a large pharmacological study, Zimmermann et al. showed that many drugs could be metabolized by specific members of the GM; in particular, DXM, prednisone, prednisolone, cortisone and cortisol were metabolized by C. scindens and Propionimicrobium lymphophilum into androgens. Mice colonized with C. scindens exhibited lower concentrations of DXM in their caecum and higher concentrations of androgens than germ-free mice [START_REF] Zimmermann | Mapping human microbiome drug metabolism by gut bacteria and their genes[END_REF] . Because androgens (and estrogens) are also metabolized by some gut microbiota members (Steroidobacter denitrificans [START_REF]Anaerobic and aerobic cleavage of the steroid core ring structure by Steroidobacter denitrificans[END_REF] and Comamonas testosterone [START_REF] Zhang | Cloning and characterization of a novel β-ketoacyl-ACP reductase from Comamonas testosteroni[END_REF] ), the net clinical consequences of these processes on the intestinal androgens are challenging to investigate. Among the hypothetical consequences of an increase in androgens due to GM-metabolized prednisone are: prostate cancer [START_REF] Ly | Bacterial steroid-17,20-desmolase is a taxonomically rare enzymatic pathway that converts prednisone to 1,4-androstanediene-3,11,17-trione, a metabolite that causes proliferation of prostate cancer cells[END_REF] , bowel disorders and mood changes [START_REF] So | Sex-bias in irritable bowel syndrome: linking steroids to the gut-brain axis[END_REF] .

Finally, in a mouse model of lupus, He et al. demonstrated that an improvement of lupus was associated with prednisone-induced GM modifications [START_REF] He | Alterations of the Gut Microbiota Associated With Promoting Efficacy of Prednisone by Bromofuranone in MRL/lpr Mice[END_REF] . These included decreases in Mucispirillum, Oscillospira, Bilophila and Rikenella populations, and an increase in Anaerostipes.

In conclusion, GCs are responsible, at least in part, for the modification of the intestinal barrier. The mechanisms underlying GC-induced GM modifications need further investigation but could involve the disruption of the circadian clock, and the modulation of the immunology of the intestinal barrier. GM also appears to be an actor of the metabolism of GCs (Figure 2).

Calcineurin inhibitors

Tacrolimus

Tacrolimus is a macrolide that was first developed as an antibiotic before the discovery of its immunosuppressive properties. Tacrolimus is a calcineurin inhibitor that binds to the FK506binding protein to form a complex that inhibits calcineurin phosphatase [START_REF] Thomson | Mode of action of tacrolimus (FK506): molecular and cellular mechanisms[END_REF] . For over ten years it has been the cornerstone of anti-rejection treatment in kidney transplantation [START_REF] Ekberg | Reduced Exposure to Calcineurin Inhibitors in Renal Transplantation[END_REF] . Despite its reliable efficacy in maintaining immunosuppression and avoiding graft rejection, tacrolimus exhibits major metabolic side effects including glucose intolerance, diabetes [START_REF] Heisel | New Onset Diabetes Mellitus in Patients Receiving Calcineurin Inhibitors: A Systematic Review and Meta-Analysis[END_REF] and high blood pressure [START_REF] Hoorn | Pathogenesis of calcineurin inhibitor-induced hypertension[END_REF] .

Tacrolimus modifies the gut microbiota

During the past five years, several studies have demonstrated an impact of tacrolimus on the rodent fecal microbiota (Figure 3). While these studies consistently identified changes in the GM, the characteristics of the changes diverge.

When given by oral gavage, tacrolimus did not alter the richness (alpha diversity) the Firmicutes/Bacteroidetes ratio [START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF][START_REF] Bhat | Impact of Immunosuppression on the Metagenomic Composition of the Intestinal Microbiome: a Systems Biology Approach to Post-Transplant Diabetes[END_REF][START_REF] Zhang | Immunosuppressive effect of the gut microbiome altered by high-dose tacrolimus in mice[END_REF] , neither in rats nor in mice. However, Toral et al. found that intraperitoneal injection of tacrolimus in a mouse model was responsible for a decrease of microbial diversity as measured by the Shannon Index, and an increase of the Firmicutes/ Bacteroidetes ratio [START_REF] Toral | Lactobacillus fermentum Improves Tacrolimus-Induced Hypertension by Restoring Vascular Redox State and Improving eNOS Coupling[END_REF] .

In rats, intraperitoneal tacrolimus decreased the fecal relative abundance of Mollicutes, Micrococcaceae, Actinomycetales, Roseburia, Oscillospira, Rothia, and Staphylococcus and increased A. muciniphila [START_REF] Bhat | Impact of Immunosuppression on the Metagenomic Composition of the Intestinal Microbiome: a Systems Biology Approach to Post-Transplant Diabetes[END_REF] . In mice, oral tacrolimus decreased Ruminococcaceae (of which Oscillospira and Ruminococcus 87 ), Clostridium, Rikenella, and Bifidobacterium 88 and increased the abundance of Allobaculum, Bacteroides, Lactobacillus and A. muciniphila.

These variations of the intestinal microbiota are relevant for several reasons: first, Allobaculum was found to be increased in a model of immunodeficient mice [START_REF] Dimitriu | Temporal stability of the mouse gut microbiota in relation to innate and adaptive immunity[END_REF] and might be a "gut signature" of tacrolimus-induced immunosuppression. Second, tacrolimus triggers a gut dysbiosis that is analogous to that observed in metabolic diseases, i.e. an increased Firmicutes/Bacteroidetes ratio that is a major marker of the gut dysbiosis linked to metabolic diseases [START_REF] Turnbaugh | A core gut microbiome in obese and lean twins[END_REF][START_REF] Ley | Obesity alters gut microbial ecology[END_REF][START_REF] Turnbaugh | An obesity-associated gut microbiome with increased capacity for energy harvest[END_REF] . Moreover, tacrolimus seems to have a depleting effect on short-chain fatty acid producing bacteria such as Ruminococcus spp. and Bifidobacterium. Interestingly, acetate and butyrate are involved in the regulation of blood pressure [START_REF] Pluznick | Microbial Short-Chain Fatty Acids and Blood Pressure Regulation[END_REF] , lipogenesis and blood glucose 94 , [START_REF] Qin | A metagenome-wide association study of gut microbiota in type 2 diabetes[END_REF] .

Oscillospira, also depleted in tacrolimus-treated rats and mice, is associated with a "metabolic-friendly" microbiota [START_REF] Konikoff | Oscillospira: a Central, Enigmatic Component of the Human Gut Microbiota[END_REF] . Finally, tacrolimus-induced gut dysbiosis shares similarities with a type 2 diabetes GM that is enriched in A .muciniphila and depleted in Roseburia [START_REF] Qin | A metagenome-wide association study of gut microbiota in type 2 diabetes[END_REF] .

However, it is possible that tacrolimus has different impacts on the GM depending on the dose that was administered. In a rat liver transplant model, an intermediate dose (0.5 mg/kg of body mass) of tacrolimus was associated with an increase of microbial richness and of the beneficial bacteria Bifidobacterium and F. prausnitzii, and a decrease of less beneficial bacteria such as Enterobacteriaceae and Bacteroides-Prevotella. These changes were associated with a better liver transplant outcome. In contrast, lower (0.1 mg/kg) and higher (1 mg/kg) doses of tacrolimus were associated with better microbial richness, and an increased abundance of Enterobacteriaceae and decrease of Bifidobacterium and F. prausnitzii. Both high and low doses of tacrolimus were associated with a poorer allograft outcome and endotoxinemia [START_REF] Jiang | Optimal immunosuppressor induces stable gut microbiota after liver transplantation[END_REF] .

Tacrolimus alters the GM metabolic functions

Using PICRUST analysis, a bioinformatic tool that predicts the functional composition of the GM using marker gene data [START_REF] Langille | Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences[END_REF] , tacrolimus was found to alter functions of the GM such as lipid and carbohydrate metabolism [START_REF] Zhang | Immunosuppressive effect of the gut microbiome altered by high-dose tacrolimus in mice[END_REF] . These results were confirmed by metagenomic analysis [START_REF] Bhat | Impact of Immunosuppression on the Metagenomic Composition of the Intestinal Microbiome: a Systems Biology Approach to Post-Transplant Diabetes[END_REF] .

Tacrolimus altered carbohydrate metabolism and starch degradation by inducing a shift from a mostly anabolic microbiota towards a catabolic one [START_REF] Bhat | Impact of Immunosuppression on the Metagenomic Composition of the Intestinal Microbiome: a Systems Biology Approach to Post-Transplant Diabetes[END_REF] .

Interestingly, modulation of the GM with a strain of Lactobacillus that is used as a probiotic prevented tacrolimus-induced hyperglycemia in rats [START_REF] Bhat | Impact of Immunosuppression on the Metagenomic Composition of the Intestinal Microbiome: a Systems Biology Approach to Post-Transplant Diabetes[END_REF] . Toral et al. [START_REF] Toral | Lactobacillus fermentum Improves Tacrolimus-Induced Hypertension by Restoring Vascular Redox State and Improving eNOS Coupling[END_REF] showed that fecal microbiota transplantation from tacrolimus-treated mice induced high blood pressure in the recipients. Another strain of Lactobacillus (LC40) also corrected tacrolimus-induced high blood pressure and endothelial dysfunction.

Tacrolimus alters the intestinal barrier and modulates immunity

Tacrolimus increases intestinal permeability in a dose-dependent manner (Figure 3). This could be a consequence of a decreased expression of occludin and Muc3 in the colon [START_REF] Toral | Lactobacillus fermentum Improves Tacrolimus-Induced Hypertension by Restoring Vascular Redox State and Improving eNOS Coupling[END_REF] and partly responsible for the observed endotoxinemia [START_REF] Jiang | Optimal immunosuppressor induces stable gut microbiota after liver transplantation[END_REF] . Moreover, tacrolimus is responsible for a local immunosuppression in the gut by inhibiting mucosal T-lymphocyte and NK cells functions [START_REF] Van Dieren | Local Immune Regulation of Mucosal Inflammation by Tacrolimus[END_REF] . Tacrolimus also decreases RegIIIβ and RegIIIɣ expression [START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF] , two lectins (antimicrobial peptides secreted by the host as a response to IL-22) that regulate bacterial density in the GM and are involved in the host's innate immunity response to the GM [START_REF] Muniz | Intestinal antimicrobial peptides during homeostasis, infection, and disease[END_REF] . This in turns possibly modifies the GM [START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF] .

Interestingly, either tacrolimus treatment or the transfer of the fecal microbiota from tacrolimus-treated mice resulted in an increase of the proportion of CD4 + CD25 hi FoxP3 + regulatory T cells in the colonic mucosa of recipient mice, as well as the circulation [START_REF] Zhang | Immunosuppressive effect of the gut microbiome altered by high-dose tacrolimus in mice[END_REF] . This is a clear indication that the effect of tacrolimus on regulatory T cells is mediated through the microbiota.

Using a skin transplant model, Zhang et al. demonstrated that fecal transfer from tacrolimustreated mice elicited immunosuppressive properties, and improved skin allograft survival in the recipient when combined with a low-dose of tacrolimus, administered by gavage 87 .

Gut microbiota: an actor of tacrolimus metabolism

A pilot study 100 has shown that patients who require high doses of tacrolimus to reach the target trough level (plasma concentration) harbored a higher relative abundance of F.

prausnitzii in their GM. Moreover, F. prausnitzii abundance at one week after KT was positively correlated with future dosing of tacrolimus at one month. This study provides evidence for a new pathway of tacrolimus metabolism, even if some confounding parameters

were not taken into account (such as patients dietary habits that modulate both tacrolimus absorption 101 and F. prausnitzii abundance, or CYP3A5 gene polymorphism, which is known to modulate FK pharmacokinetics 102 ) this study possibly evidences a new pathway for tacrolimus metabolism.

The same authors 103 showed that some commensal gut bacteria such as F. prausnitzii or Clostridiales were able to transform tacrolimus into a 15-fold less active metabolite in vitro.

To summarize, tacrolimus alters the GM composition which affects the host. The GM of tacrolimus-treated mice seems to have immunosuppressive properties. Even if the results are not always consistent, tacrolimus appears to deplete bacteria that positively affect their host's metabolism. These results question the role of the GM in tacrolimus' immunosuppressive activity and side effects. An additional level of complexity comes from the observation that the response of the GM may differ depending on the dose of tacrolimus. Conversely, the GM could be involved in tacrolimus metabolism and explain part of the inter-patient variability of tacrolimus dosing.

Cyclosporine

Cyclosporine

4.

Anti-metabolites 4.1.

Mycophenolate Mofetil

Mycophenolate Mofetil (MMF) is a prodrug that is converted into mycophenolic acid (MPA), which inhibits purine synthesis through the inhibition of inosine monophosphate dehydrogenase (Figure 4). After intestinal uptake MPA is glucuronidated by a liver enzyme, leading to its inactivation. The inactive form of MPA is then eliminated by the bile and the stool 106 .

It is the main drug responsible for digestive disorders after KT, affecting 30 to 50% of all MMF-treated patients 107 with symptoms that include diarrhea, abdominal pain, vomiting, and, rarely, digestive ulcer or haemorrhage 107 . Endoscopic and histological patterns of MMFinduced colitis can mimic acute colitis 108 . This major side effect can lead to an impaired quality of life and an irregular observance, increasing the risk for allograft rejection 109

The pathophysiology of MMF-induced colitis has recently been explored and there is evidence for a potential role of the GM.

MMF induces gut microbiota modification and endotoxemia.

Mice treated with MMF exhibited significant markers of dysbiosis, such as an early and persistent diminution of microbial richness, an increase of the Firmicutes/Bacteroidetes ratio and a relative abundance of Clostridia and Bacteroides spp. 110 and of Proteobacteria, including the opportunistic pathogens Escherichia and Shigella. In contrast Akkermansia, Parabacteroides and Clostridium were decreased 111 .

These abnormalities were associated with an increase of circulating lipopolysaccharide (LPS)

, a major cell wall component of Gram-negative bacteria. Endotoxemia leads to low-grade inflammation that in turn promotes metabolic syndrome and insulin resistance in rodents 112 , and cardiovascular events in KT recipients 113 . This increased endotoxemia is attributed to the increase of Gram-negative bacteria in the GM and an alteration of gut tight junction induced by MPA, resulting in increased gut permeability 114 .

MMF-induced colitis is associated with dysbiosis.

In conventionally raised mice, MMF induces cecal atrophy, colitis and weight loss 110 . When MMF was administered to axenic mice, or mice previously treated with broad-spectrum antibiotics, they did not develop these disorders, suggesting that MMF induces a dysbiosis that is responsible for the digestive disorders.

Taylor et al. showed that MMF was responsible for an increase of Clostridia, and Bacteroides spp. in mice 110 . They observed a simultaneous rise in gene expression and activity of gut Bglucuronidase (GUS) in the caecum and the colon. This enzyme, which is expressed by Bacteroides, converts the inactive glucuronidated MPA into its free active form. Interestingly, addition of vancomycin was responsible for a decrease of Bacteroides, of GUS activity and of free MPA in mice's stools. The use of vancomycin also abrogated MMF gastro-intestinal side effects such as weight loss, cecal atrophy and colonic inflammation. Moreover, in eleven human patients, the author found that GUS activity correlated with MMF exposure. Finally, intrarectal infusion of MPA was responsible for weight loss in mice. Altogether, these results suggest that MMF promotes Bacteroides proliferation in the gut of mice, which results in an increased GUS activity and therefore an increase of colitogenic free MPA in the colon.

These two studies therefore provide interesting insights into the pathophysiology of MMFinduced gastro-intestinal side effects.

Azathioprine

Azathioprine (AZA) is another anti-metabolite that has been used for a long time in kidney transplants 115 . It is the prodrug of 6-mercaptopurine, a purine antagonist which inhibits DNA synthesis 116,117 .

AZA was found to inhibit the proliferation of some enteric bacteria in vitro: Campylobacter concisus, Bacteroides fragilis, and Bacteroides vulgatus 118 . Only the highest concentration of AZA (200 µg/ml) inhibited the growth of E. coli. AZA did not significantly affect the growth of E. faecalis. In a cohort of 20 patients with inflammatory bowel diseases, AZA was found to increase the concentration of mucosal bacteria compared to healthy controls, and the percentage of the epithelial surface covered with adherent bacteria compared to patients with inflammatory bowel diseases 119 .

mTOR inhibitors

Rapamycin and everolimus are mammalian target of rapamycin (mTOR) inhibitors which interfere with lymphocyte proliferation 120 by inhibiting mTOR complex 1 and 2. They are frequently used in SOT and are responsible for metabolic, infectious and hematologic adverse events 121 . Recent studies have highlighted the impact of this therapeutic class on the GM.

Rapamycin and everolimus change gut microbiota composition

Everolimus seems to have little impact on the GM [START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF] . The global composition was not significantly altered in mice treated with this drug as compared to controls as evaluated by principal component analysis. Rapamycin decreased the phylogenetic diversity score in rats [START_REF] Bhat | Impact of Immunosuppression on the Metagenomic Composition of the Intestinal Microbiome: a Systems Biology Approach to Post-Transplant Diabetes[END_REF]

Effect of mTOR inhibition on gut barrier function and immune and metabolic disorders.

Intestinal cell differentiation and proliferation are regulated by mTOR Complex 1, in a Notchdependent manner. The excessive activation of this pathway alters goblet and Paneth cell differentiation [START_REF] Hill | Intestinal Bacteria and the Regulation of Immune Cell Homeostasis[END_REF]124 . This stimulation is attenuated by rapamycin 125 . Paneth cells, located in the small intestinal mucosa, play a central role in the crosstalk between the innate immune system and the GM through the synthesis of antimicrobial peptides. We showed that, in mice, everolimus decreases the ileal expression of IL-

Belatacept

In 2011, belatacept was approved as a maintenance immunosuppressive drug after kidney transplantation in 2011 and shows better patient and graft survival, and better kidney function No data regarding the impact of this treatment on the GM has been published so far.

However, a recent case report relates the occurrence of severe intestinal lesions (Crohn-like colitis) induced by belatacept 131 . The possible role of the GM in this complication needs to be explored.

Alemtuzumab

Alemtuzumab is a monoclonal antibody that binds to the CD52 antigen, thereby depleting T and B-lymphocytes 132 . In KTs, alemtuzumab is used for induction, and efficiently reduces post-transplant acute rejection 133 .

Alemtuzumab alters the GM and mucosal immunity

In addition to its depleting effect on circulating lymphocytes, alemtuzumab also depleted and altered the function of intestinal mucosa lymphocytes in a monkey model 134 . This depletion of T-lymphocytes in intestinal mucosa was associated with a shift in the GM composition. In ileal mucosa, alemtuzumab was responsible for an increase of Prevotella, and a higher proportion of opportunistic pathogenic bacteria such as E. coli, Shigella Flexneri. In the fecal microbiota, alemtuzumab was responsible for an increase of Clostridiales and a decrease of F.

Prausnitzii. Interestingly, microbial modifications were restored 35 days after alemtuzumab discontinuation and mucosal lymphocyte regeneration.

Alemtuzumab alters the fungal microbiota.

Mucosal lymphopenia induced by alemtuzumab was also associated with fungal microbiota dysbiosis in a cynomolgus monkey model 135 . There was an increased diversity and colonization by Candida albicans, Aspergillus clavatus, and Saccharomyces cerevisiae.

Interestingly, resilience of the fungal microbiota was observed in this study, as the fungal microbiota of colonic mucosa was restored concomitantly with T lymphocyte reconstitution.

Alemtuzumab treatment decreased the diversity of the fecal fungal microbiota, and increased Candida albicans, Saccharomyces cerevisiae and Botryotinia fuckeliana.

Combined therapy

Very few studies have investigated the impact of simultaneous multiple IST on the GM (Figure 5) even though this is clinically relevant, because ISTs in transplantation are in fact usually combined 136 , as per recommendations 137 .

Rodent studies

We have shown that a combined therapy including prednisolone, tacrolimus and MMF depleted Clostridium sensu stricto in the ileum. The combined therapy also altered the GM, by modifying the global composition of the fecal microbiota [START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF] , favoring the proliferation of both commensal and uropathogenic E.coli. We hypothesized that this microbiological side effect could participate in the high prevalence of urinary tract infections after KT 138 .

It is possible that the decreased secretion of innate ileal anti-microbials (lectins RegIIIɣ and RegIIIβ) participated in these modifications. Lectins were also decreased after treatment with prednisolone and tacrolimus separately, but no synergistic action of the combined treatment was observed compared to monotherapies.

Human studies.

In a study that included a small number of patients, Zaza et al. have demonstrated that KT recipients treated with tacrolimus and MMF exhibited a different gut composition in bacterial genes categorized by function than patients treated with everolimus and MMF 139 . Treatment with tacrolimus was associated with a decrease in the genes coding for the macrolide transport system (mrsA) and an enrichment in genes coding for flagellar motor switch proteins and type IV pilus assembly proteins. These proteins are expressed by Enterobacteriaceae and the bulk enrichment in genes coding for the flagellar apparatus could be the consequence of their proliferation under immunosuppressed conditions. Thus, this study suggests the development of a specific gut signature in IST-treated patients with specific functional characteristics, the clinical implication of which still have to be explored.

Direct and indirect alterations of the GM by IST

It appears that there are bidirectional interactions between the GM and the IST-modified host immunity. A key question is to decipher the underlying mechanisms of this crosstalk. This an extremely arduous task as there are many "speakers" partaking in this dialogue. The GM communicates through metabolites and antigens. The immune system detects these signals (microbial-associated molecular patterns) through receptors such as toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NOD-like receptors; NLRs), and reacts through innate (antimicrobial peptides, protein of the mucus) and adaptive immunity (both cellular and humoral) responses. The result is a complex, highly multifactorial, and adaptive network of consequences which favor some bacterial species and disadvantages others. IST can interfere with basically all the components cited here and it is very complicated to prove that the decrease (or the increase) of one specific bacterial group is the direct consequence of one immunological pathway. In this paragraph, we provide succinct tracks to explore the topic.

The direct effect of IST on bacterial growth

Many ISTs are antimicrobial in nature. Tacrolimus was first developed as an antibacterial and also has a direct antifungal activity. Thus, tacrolimus may alter the microbial gut balance.

Similarly, rapamycin is a macrolide derived from Streptomyces hygroscopicus that is also effective against fungi 140 . However, to our knowledge, the direct effect of IST on specific GM components has not been studied. This is because the culture of many gut bacteria species remains challenging. Furthermore, the in vivo antimicrobial activity of IST probably depends on pharmacological parameters that are difficult to explore, including the difficult-to-establish IST drug concentrations in the various components of the digestive tract: mucosa, mucus and intestinal lumen. Mucus-associated GM communities are probably not affected in the same way by IST than communities residing in the lumen. Recently, Jalili-Firoozinezhad et al. were able to establish a complex human GM culture in vitro 141 . This will be a critical tool to explore the direct effect of IST (and other treatments) on the GM.

Indirect effects of ISTs on the GM through the modification of host's immunity

Controlling GM overgrowth and containing bacteria within the digestive lumen (compartmentalization) are essential to the health of the host. As an example, Myd88 -/-mice develop to harbor commensal bacteria in their spleen [START_REF] Slack | Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism[END_REF] . Furthermore, to ensure an adequate compartmentalization, both innate and humoral immunities are necessary. Indeed, Myd88 -/- and Ticam -/-double-deficient mice not only harbor commensal mice in their spleen, but also spontaneously develop high titers of anti-commensal serum IgGs [START_REF] Slack | Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism[END_REF] . The control of the immune system over the GM has been extensively reviewed by Willing et al. 142 . As various IST can alter the gut mucus [START_REF] Wu | Chronic glucocorticoid treatment induced circadian clock disorder leads to lipid metabolism and gut microbiota alterations in rats[END_REF][START_REF] Huang | Using corticosteroids to reshape the gut microbiome: implications for inflammatory bowel diseases[END_REF] , the global production of immunoglobulins, neutrophils (producing steroids and MMF) and T-and B-cell function, it is probable that ISTs modify the GM indirectly through immunomodulation. In favor of this hypothesis we have found that prednisolone or a combination of prednisolone, tacrolimus and MMF significantly reduces the production of ileal IL-22 resulting in a decreased expression of the gut C-type lectins Reg3γ

and Reg3β, two anti-microbial peptides that directly affect the GM [START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF] . Lastly, we have also shown that an IST comprising with prednisolone, tacrolimus and MMF is responsible for the proliferation of both commensal and extra-intestinal pathogenic E. coli [START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF] . It is very unlikely that this bacterial proliferation is a direct consequence of IST on E. coli, rather than an indirect consequence through immunomodulation.

Limitations

Variability of studies on rodent GM.

The different studies on the impact of IS drugs on the GM show similarities but also differences, making it difficult to draw a reproducible and reliable phenotype of IS-induced gut dysbiosis.

Many factors contribute to this heterogeneity of results. Indeed, the GM is highly variable and many environmental factors can modulate its composition. Among these are chow composition, husbandry ventilation, barrier access, day/light cycle, and other animal facility parameters [143][144][145] .

Some intrinsic factors may also explain inter-study microbiota variation such as rodent age or rodent strain 146,147 . Laboratory techniques for DNA extraction and microbiota analysis also influence results 148 . Some authors have suggested a quality control procedure to homogenize research on the GM, in order to increase its reproducibility 149 . It is also important to consider that IST-induced effects probably depend on the initial composition of the GM. We have shown that the effect of IST on the GM differed between two identically designed replicates of an experiment probably because of a different initial microbiota composition [START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF] .

Finally, it is probable that the effects of IST on the GM differ according to the dose used in each study [START_REF] Jiang | Optimal immunosuppressor induces stable gut microbiota after liver transplantation[END_REF] .

Extrinsic validity of rodent GM studies.

Husbandries are required to maintain strict hygienic rules to avoid sanitary disasters. As a consequence, mice raised in these aseptic environments exhibit differences in their gut, vaginal and skin microbiota, and in their immune systems compared to "dirty mammalians"

150 . For that reason, the generalization to wild mice is limited. Interventions such as the "wilding" of laboratory mice could decrease the physiologic and metagenomic gap between laboratory rodents and free-ranging mammals including humans 150 .

Conclusions.

The GM is altered after SOT and these modifications, both before and after transplant are associated with post-transplantation adverse events. IST reshapes the GM and alter its structural and functional composition. Because IST-induced dysbiosis might contribute to these adverse events, a better understanding of the genesis of this dysbiosis is necessary.

On the one hand IST-induced dysbiosis probably participates in the immunosuppressive effects of these treatments and could improve allograft survival. On the other hand, alterations of the GM may be linked to IST-related adverse events such as metabolic disorders, colonic inflammation or intestinal colonization by pathogens.

ISTs deeply affect the intestinal barrier, from the mucus secretion to mucosal immunity 151 and intestinal permeability. Another level of complexity is that IST-induced gut barrier alterations have been shown to be both a cause and a consequence of GM modification. The depletion of the microbiota associated with the weakening of the intestinal barrier could promote the colonization of pathogenic bacteria observed in several studies. Finally, GM also modulates IS drug metabolism which probably participates to the inter-patient variability of IST dosing to reach therapeutic trough levels.

A better understanding of GM implication in SOT could thus help clinicians to prevent the occurrence of post-transplant adverse outcomes.

In the future, modulation of the intestinal microbiota, either by the use of probiotics or by the transfer of fecal microbiota could help prolong the survival of the graft and limit the occurrence of side effects related to immunosuppressive treatments.

  A (CSA) is another calcineurin inhibitor used in SOTs. It selectively inhibits Tlymphocyte activation 104 . Jia et al. studied the impact of CSA on the GM in a rat model of allogenic liver transplantation 105 . The GM was explored by denaturing gradient gel electrophoresis and by the quantification of specific bacteria using PCR. CSA increased the GM richness with an enrichment of F. prausnitzii compared to the control group (allograft without CSA). On the contrary, CSA decreased the proportion of Enterobacteriaceae and of Clostridium clusters I and XIV. Interestingly, the analysis of the GM gel electrophoresis profiles clustered the non-rejecting rats (sham surgery, isograft and CSA-treated allograft) as opposed to the rejecting rats (non-treated allograft). This indicates that CSA tends to restore the pre-transplant GM.

  in the long term compared to CSA based regimens 129 . It blocks T-cell activation by inhibiting the co-stimulation signal 130 .

Figure 1 :

 1 Figure 1: Factors which affect the intestinal microbiota after kidney transplantation.These can be divided in pharmacological factors, such as anti-infectious treatments[START_REF] Modi | Antibiotics and the gut microbiota[END_REF] , immunosuppressive drugs (this review) and anesthetics 152 , and non-pharmacological factors, such as the normalization of renal function and associated metabolic abnormalities 124 , the modification of dietary habits 153 and the discontinuation of chronic hemodialysis 154

Figure 2 :

 2 Figure 2: Impact of glucocorticoids (GCs) on the gut microbiota (GM). GCs alter the gut microbiota composition at different phylogenic levels depending on the studies, the dosing and the host (patients, or rodent model). Under physiological conditions, GCs inhibit the expression and synthesis of Muc2, the main component of colonic mucus. GCs also alter gut immunity: first, they downregulate the ileal expression of antimicrobial lectins RegIII β and Reg III ɣ, via the inhibition of IL-22; second, they restrict the coating of bacteria by mucosal IgA. On the other hand, GCs induce a retightening of TNF-α-induced tight junction relaxation by downregulating myosin light chain kinase (MLCK) synthesis and Myosin Light Chain 2 (MLC2) phosphorylation. These modifications of the gut barrier may cause GM modifications. The dysregulation of the circadian clock by exogenous GCs could also result in gut dysbiosis. In mice, GCs delay the restoration of the GM after antibiotic treatment, exposing the animals to more severe Clostridium difficile infection. Finally, C. scindens converts GCs into androgens, implicating the GM in the metabolism of GCs. GCs: glucocorticoids. GM: gut microbiota. Muc2: mucin 2. MLC2: Myosin Light Chain 2. MLCK: MLC Kinase. Some elements of the Figure were obtained from Servier Medical Art®.

Figure 3 :

 3 Figure 3: Impact of tacrolimus on the gut microbiota. Tacrolimus-induced dysbiosis results in functional alterations of the GM. Tacrolimus confers immunosuppressive properties to the GM both at the local and the systemic levels by increasing the population of Treg lymphocytes. Tacrolimus-induced GM alterations could also result in some of the drug's side effects such as high blood pressure and diabetes. Tacrolimus increases the gut permeability

Figure 4 :

 4 Figure 4: Impact of mycophenolate mofetil (MMF) on the gut microbiota. MMF strips the diversity of the GM, increases the ratio Firmicutes/Bacteroidetes, and favors the Proteobacteria, a phylum that harbors pathogenic strains such as Shigella and E. coli. This gut dysbiosis generates high fecal concentrations of lipopolysaccharides (LPS) and colonic inflammation. In addition, mycophenolic acid (MPA), the active metabolite of MMF, perturbs tight junctions by upregulating Myosin Light Chain Kinase (MLCK) and Myosin Light Chain 2 (MLC2) phosphorylation. Combined, this is responsible for endotoxemia, a phenomenon that is associated with a higher rate of cardiovascular events after KT. Finally, the abundance of Bacteroides sp. correlates with a high level of activity of colonic bacterial β-glucuronidase, an enzyme that converts the glucuronated form of MPA (MPAG) back into its active form. Modulation of the GM with antibiotics reduces β-glucuronidase activity, decreases colonic MPA levels and amends MMF's digestive side effects. MMF: mycophenolate mofetil. GM: gut microbiota. LPS: lipopolysaccharides. KT: kidney transplant. MPA: mycophenolic acid. MLC2: Myosin Light Chain 2. MLCK: MLC Kinase. Some elements of the Figure were obtained from Servier Medical Art®.

Figure 5 :

 5 Figure 5: Impact of combined therapy on the gut microbiota. A combination of tacrolimus, MMF and prednisolone promotes both commensal and extra-intestinal pathogenic E. coli proliferation and decreases the ileal relative abundance of Clostridium sensu stricto. Combined therapy decreases ileal gut immunity by decreasing IL-22, RegIIIβ and RegIIIɣ expression. MMF: mycophenolate mofetil. Some elements of the Figure were obtained from Servier Medical Art®.

Figure 6 :↗

 6 Figure 6: Symbols used in the Figures

  , and influenced beta diversity but not alpha diversity in mice 122 . Specifically, rapamycin affected gut dysbiosis induced by a high fat diet i.e. it changed the relative abundance of Helicobacter, and Coprobacillus. In a rat model, rapamycin decreased the bacterial diversity and altered beta diversity composition with a decrease of Roseburia, Oscillospira, Mollicutes, Rothia, Micrococcaceae, Actinomycetales, and Staphylococcus[START_REF] Bhat | Impact of Immunosuppression on the Metagenomic Composition of the Intestinal Microbiome: a Systems Biology Approach to Post-Transplant Diabetes[END_REF] .Using a Drosophila model, Schinaman et al. found that rapamycin treatment was associated with alteration of the gut microbiota, namely a reduction of bacterial mass and decrease of

	Turicibacter,	unclassified	Marinilabiliaceae,	Alloprevotella,	unclassified
	Porphyromonadaceae, Ruminococcus, Bifidobacterium, Marvinbryantia, Ruminococcus
	(Lachnospiraceae), Alphaproteobacteria 123

  22 and consequently of C-type lectins RegIIIβ and RegIIIɣ[START_REF] Tourret | Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli[END_REF] . The mTOR pathway is an important actor in intestinal epithelial cell proliferation, through Wnt signaling. Therefore, inhibition of mTOR interferes with enterocyte proliferation126 . In a murine model, Xie et al. found that hyperactivation of intestinal mTOR induced by a Western diet was responsible for an increased necroptosis of intestinal epithelial cells, and a marked susceptibility to develop colitis 127 . It was associated with gut barrier dysfunction, measured by an increased intestinal permeability (an increased blood translocation of FITC-Dextran given by gavage). Rapamycin prevented colitis and reduced inflammation. Furthermore, GM depletion by antibiotics attenuates mTOR

	hyperactivation associated with intestinal disorders, further suggesting that the GM is
	involved in mTOR hyperactivation. 127
	Finally, Rapamycin-induced gut dysbiosis correlated with metabolic disorders such as body
	weight increase, insulin resistance, intestinal inflammation (measured through fecal lipocalin-
	2) and fat deposition in mice receiving a high fat diet 122 . Peyer patch immune modifications
	(increases in IL17 + CD4

+ T cells) induced by rapamycin correlated with GM modifications 128 .

Acknowledgments / funding

None

There was no external funding for the preparation of this review

Data availability statement

Data sharing not applicable -no new data generated

Abbreviations

Disclosure

The authors have no disclosure to declare