L Arantchouk

G Point

Y Brelet

B Prade

J Carbonnel

Y.-B André

A Mysyrowicz

A Houard

Large scale Tesla coil guided discharges initiated by femtosecond laser filamentation in air

The guiding of meter scale electric discharges produced in air by a Tesla coil is realized in laboratory using a focused terawatt laser pulse undergoing filamentation. The influence of the focus position, the laser arrival time or the gap length is studied to determine the best conditions for efficient laser guiding. Discharge parameters such as delay, jitter and resistance are characterized. An increase of the discharge length by a factor 5 has been achieved with the laser filaments, corresponding to a mean breakdown field of 2 kV/cm for a 1.8 m gap length. Consecutive guided discharges at a repetition rate of 10 Hz are also reported.

I. INTRODUCTION

Intense ultrashort laser pulses propagating through atmosphere give rise to spectacular non-linear effects. Owing to a dynamic competition between non-linear optical Kerr self-focusing, diffraction, multiphoton absorption and plasma induced defocusing, a contracted beam of high peak intensity can be formed over long distances. This intense laser pulse leaves in its wake a long and thin column of weakly ionized plasma. This process is usually referred to as filamentation [1][2][3][4]. Plasma filaments are useful for remote manipulation of high voltage (HV) discharges. They can trigger and guide MV discharges over several meters [5][6], carry high DC currents with reduced losses [7] or deviate arcs from their natural path [8]. These properties are of great interest for applications such as the laser lightning rod [START_REF] Zhao | Ultrafast Phenomena[END_REF][10], virtual plasma antennas for radiofrequency (RF) transmission [11][12], plasma aerodynamic control [13][14], high voltage switch with low jitter [15][16], or compact Marx generator [17].

Experiment on triggering and guiding of large scale (0.5 -3 m) discharges by use of laser-induced ionized filament have been performed so far with monopolar pulsed or DC high voltage sources, where the electric field direction remains constant, typically with Marx generators [18][19], or Van de Graaf machines [20]. It has been shown that the presence of the plasma filament can decrease the breakdown voltage by 30 % to 50 % depending on the voltage polarity for a 2.5 m spark with a Marx generator [8].

Recently, demonstration of discharge triggering and guiding by femtosecond laser filaments using a Tesla coil generator has been reported by two groups revealing a large reduction of the AC breakdown field with the filaments [21][22][23]. In this manuscript we study in detail the influence of the different experimental parameters on the guiding efficiency and on the spark parameters. We observe that the guiding effect strongly depends on the amplitude and polarity of the Tesla field when the filament is formed between the electrodes.

II. METHODS

A. Experimental setup

The ENSTAmobile laser was used to create the plasma filaments in air. This Chirped Pulse Amplified (CPA) laser chain can deliver 50 fs pulses with energy of 300 mJ at a maximum repetition rate of 10 Hz. For the present experiment, the 50 fs pulses were chirped to 700 fs. The laser beam was focused in air by a 5 m converging lens, generating a bundle of multifilaments extending 200 cm before and 50 cm after the geometrical focus of the lens. A detailed analysis of the generated filaments can be found in ref [24]. Two brass spherical electrodes (ø 10 mm), one linked to the Tesla coil, the other to the ground, are used to create the discharge. They were aligned with the laser beam so that filaments skimmed both of them. In usual experimental geometry the laser beam met the HV electrode first.

B. Description of the Tesla coil

a) Author to whom correspondence should be addressed. Electronic mail: aurelien.houard@ensta.fr.

The Tesla resonant transformer consists of two magnetically coupled LC circuits with close resonant frequencies Ω 0 = (L 1 C 1 ) 1/2 = (L 2 C 2 ) 1/2 ≈ 100 kHz (cf. Figure 1). The primary capacitor C 1 is made of twenty parallel ceramic disc capacitors (2 nF each, i. e. C 1 = 40 nF). The operation of the generator is governed by a trigatron allowing synchronization of the HV source with the laser with a temporal jitter < 20 ns. The triggering of C 1 charged up to 8-14 kV generates a magnetic field in L 1 , which is coupled via mutual inductance with L 2 . The secondary coil has a diameter of 20 cm and a length of 100 cm with N = 1150 turns, which yields L 2 = 52.9 mH. The output distributed capacitor C 2 is composed of two parts, neglecting the capacitance arising from room environment. The first one is the self-capacitance of the coil (~15.3 pF) [25] and the second one is the toroidalshape termination of the Tesla with an outer diameter of 80 cm and a cross section diameter of 16 cm, resulting in 33.6 pF. The total capacitance C 2 is then estimated to be about 49 pF. This value was found to be in good agreement with the resonance frequencies of the Tesla coil.

The voltage V 2 at the output of the Tesla secondary circuit is a superposition of two damped sinusoidal oscillations of frequencies ω 1,2 = 0.5(ω + ±ω -), with ω +/-= Ω 0 (1±k) -1/2 , where k is the effective coupling coefficient between primary and secondary coil inductances L 1 and L 2 . It results in the generation of an oscillating high electric potential also referred as a beat structure (Figure 2(a)). The experiments are carried out with a fairly small HV source delivering up to V 1 = 12.5 kV to the primary Tesla capacitor C 1 which thus stores an energy E C1 ≈ 3 J. The maximal absolute amplitude of V 2 reaches 365 kV.

C. Diagnostics

Voltage on the primary capacitor C 1 was measured by a high voltage probe (PVM-1 from North Star with an 80 MHz bandwidth). The discharge current at the output Tesla generator was monitored with a current viewing resistor installed in the ground electrode. The Tesla coil output voltage was probed by a remote antenna. This antenna, a 70 mm-wide copper disk, was directed towards the output torus of the Tesla coil, and connected to a 1-M oscilloscope channel using a 12.5 m coaxial cable, thus forming a capacitive divider. The HV part of this divider consists of the very small capacitor between the disk and the Tesla coil. Its low voltage part lies in the cable capacitance C cable = 0.1 nFm -1 x12.5 m = 1.25 nF. This approximation is valid for a time scale τ << 1.25 nF x 1 M = 1.25 ms, and can then be applied for the monitoring of the 100 kHz oscillation of the generator. The signal generated by the antenna, placed at 2.5 m from the Tesla coil, is displayed in Figure 2(a).

The calibration of the antenna was done using a resistive divider made of twenty 18-k high voltage resistances and by varying the supply voltage from 3 to 6 kV on the primary Tesla capacitor C 1 . The influence of the resistive probe on Tesla characteristics was controlled by the antenna. Despite the fact that the total resistance of the probe (360 k) was well above the Tesla output impedance Z 2 ≈ (L 2 /C 2 ) 1/2 = 33 k, the antenna signal shows about 30% drop when the probe is connected to the Tesla coil. With this correction we estimate the Tesla transformer gain G to 28.9 ± 1. This gain is very close to the theoretical value G calc = (C 1 /C 2 ) 1/2 = 28. Using this diagnostic, we estimate the precision of voltage measurements to be better than 7 %.

The experiments presented in this paper deal with the first half of the first beat period, and were done using an input voltage V 1 = 12.5 kV DC, yielding a maximum output voltage amplitude of 365 kV. The period of the high-frequency oscillation T is 9.66 µs. The antenna allows us not only to monitor the output Tesla voltage during experiments but also to indicate the moment of laser beam arrival into the inter electrode gap, as evidenced by the appearance of a noisy spike on the signal at this moment (cf.

III. RESULTS

A. Guided electric discharges

We first measured the self-breakdown voltage of the Tesla generator with our electrode configuration in absence of laser filaments. We determined that for voltage maximal amplitude of 365 kV the maximum inter-electrode spacing for which self-breakdown can occur was L max ~ 32 cm. This corresponds to an effective electric field E eff = V 2,max /L max ~ 11.4 kV/cm. The guiding effect of the filaments was characterized by means of time-integrated pictures of the discharges using a standard commercial camera (Canon EOS 650 D fitted with a 18-135 mm f/3.5-5.6 objective). Examples of such pictures are displayed in Figure 3 in the case of 32 cm-long discharges without (a) and with the presence of laser filaments in the gap (b). In the case of short gaps, close to the self-breakdown limit (40-50 cm), the inter-electrode electric field is high enough so that the guiding effect of the filaments is marginal. In this case, the path generated by the laser has to compete with naturally occurring streamers able to fill a large portion of the gap, often resulting in a partially-guided discharge, as seen on Figure 3(c) for a 40 cm gap. To ensure full guiding for such short gaps, the laser has to be sent before the inception of streamers [8]. In the case of longer gaps, as the electric field is less important, the guiding effect of the filaments dramatically improves.

FIG. 3. Time-integrated images of spark discharges: without any apparent guiding (a), fully guided (b) and partially guided by the laser filament (c). The laser pulse comes from the left and the gap length is fixed to 32 cm.

B. Influence of the position of the laser geometrical focus

First, we studied the influence of the laser beam focus position on the laser-induced breakdown probability. Five focal positions in the discharge gap were tested: on the first electrode (P1), 10 cm after this electrode (P2), in the middle of the gap (P3), 10 cm before the second electrode (P4) and finally on the furthermost electrode (P5). For a given focus position, we varied the laser arrival time t laser with respect to the Tesla triggering time, taken as t = 0. We used a 1 µs time step, ranging from t laser = 7.1 µs (Tesla voltage first negative peak) to t laser = 26.4 µs (voltage maximum negative peak). This time window is indeed considered as most favorable for breakdown because it is characterized by a rising amplitude for voltage peaks.

For the gap L = 50 cm, all tested focus positions gave breakdown probability of 100% at the negative peak 26.4 µs and positive peak 21.6 µs. With longer gaps (80 < L < 140 cm), the highest breakdown probabilities were obtained when the linear focus was positioned near the second electrode. To trigger the longest gap (L = 180 cm) we had to adjust the focus point 30 cm before the second electrode.

These results can be explained by the spatial structure of the multifilament bundle. Using impacts on photographic plates, we were able to determine that most of the filaments appear before the lens geometrical focus, and quickly disappear beyond this point. The focus position therefore needs to be carefully adapted to each experimental setup. All the results presented in this paper were obtained by focusing the laser beam on the grounded electrode, which proved to be the most adapted configuration for the studied gaps.

C. Influence of the laser arrival time

The next parameter under investigation is the breakdown probability P as a function of the laser arrival moment t laser and the gap length L. For a given L value, we again varied t laser with a 1 µs time step between t = 7 µs and t = 28 µs. Statistics are based on 10-20 shots. Two geometries were tested. In the configuration 1 the laser met the HV Tesla electrode first, then the grounded electrode. In the configuration 2, we inverted the two electrodes. Some of the data are depicted in Figure 4. For convenience, the temporal evolution of the Tesla voltage in absence of discharge is plotted on the same graphs (continuous line). First, we can see that the breakdown probability is maximum when the laser arrives close to a voltage extremum, and is low when filaments are created while the voltage is close to zero. For long gaps (L > 70 cm), voltage polarity is also an important parameter, since in configuration 1, P is maximal nearby negative voltage extrema and lower around positive voltage peaks. Opposite results arise in the inverted configuration. This polarity selectivity is less pronounced for short gaps, for which P remains high overall (Figure 4(a)). When the laser arrives early with respect to the Tesla oscillations (t laser < 15 µs), P is almost constant, losing all selectivity in time.

For very long gaps (L > 140 cm), the best results were obtained when the laser was sent on the first Tesla voltage peaks (t laser = 7.1, 11.9 and 16.7 µs) in configuration 1. We thus achieved P = 0.5 for L = 140 cm, and P = 0.06 for our limit gap L = 180 cm, corresponding to an average electric field of respectively 2.6 and 2 kV/cm. Note that electrode configuration 1 was kept for the rest of the experiments presented in this paper.

D. Breakdown delay

The breakdown delay t is defined as the time interval between the arrival of the laser pulse in the gap and the breakdown occurrence: t = t bdt laser . The influence of L and t laser on this parameter gives insight about the discharge development. Figure 5 presents the evolution of t with t laser for the two previously studied gap lengths (L = 50 and 100 cm). In the same figure are represented the positions of the Tesla voltage extrema. One can first see that breakdown events are distributed along these lines. A more careful look shows that most breakdowns take place slightly before the voltage reaches its local extremum, up to 600 ns before. For L = 50 cm, at a given t laser , breakdown events are mainly scattered in three groups, which are located around the three voltage extrema following the laser arrival. Occurrences are relatively well balanced between these groups, proving again that polarity dependence is very weak for short gaps. The delay t can be as short as 200 ns. For L = 100 cm, the three groups previously seen are reduced to one large group. Most notably, this unique ensemble is located close to the position of a negative voltage maximum, and corresponds to a delay t of at least a full period of the Tesla voltage, T. Moreover, the longer the gap, the more the time needed for the spark to develop. For very long gaps (L > 140 cm), t can be as high as 2T, that is the discharge takes as long as 20 µs to occur. This explains why the laser should be sent at early times to have the highest probability in this case.

Note that for selected parameters, very reproducible discharge can be obtained. Using a delay of 16.7 µs and gap length L = 100 cm, we were able to generate repetitive guided discharges at 10 Hz during several seconds with a 100 % probability.

E. Discharge current and spark resistance

We now study the voltage-current characteristics of the laser-triggered discharges. Typical current signals for L = 50 cm and L = 100 cm are presented in Figure 6. These curves present a main peak close to Gaussian, followed by strongly damped oscillations. Current amplitude is on the order of a few hundred amperes, and current duration is less than 100 ns. With longer gaps the main current peak tends to decrease in amplitude and to widen. As seen in Figure 2, our voltage diagnostic does not allow monitoring the voltage evolution after the breakdown due to an important electromagnetic noise. To characterize the discharge resistivity we then choose to define an operational discharge resistance R = U bd /I max , where U bd is the Tesla voltage at the breakdown time t bd and I max is the discharge current amplitude. Experimental (I max , U bd ) characteristics for short gaps are presented in Figure 7. Data corresponds to the breakdown events occurring near the main four Tesla voltage peaks (t bd = 16.8, 21.6, 26.4 and 31.2 µs). Because of insufficient statistics for the gap L = 50 cm, we joined data for both L = 40 and 50 cm. The two panels show respectively the upper right (I max , U bd > 0) and lower left (I max , U bd < 0) quadrants of the whole (I max , U bd ) plane. The Figure 7(a) shows a significant scattering for the breakdowns occurring with a positive Tesla voltage, especially when U bd is moderately high (< 340 kV). In these conditions, the generated discharges are not reproducible. We relate this fluctuating behavior to the frequent occurrence of partially-guided discharges with short gaps. Indeed, as in this case the discharge path changes unpredictably from shot to shot, so does the total plasma resistance. The scattering becomes less important as U bd increases, enabling us to estimate the discharge operational resistance from the highlighted group in Figure 7(a). This gives R = 1.2 k. As for the negative quadrant of the (I max , U bd ) characteristic, we observe no significant scattering of breakdown events (Figure 7(b)), which we cannot explain as of now. Still, to be coherent with the previous estimation, we again select the data with the highest voltage and peak current, undoubtedly resulting from full-fledged guided discharges (highlighted group in Figure 7(b)). This in turns yields an operational resistance R = 1 k.

The study of longer gaps gives characteristics such as the one displayed in Figure 7(c) for L = 100 cm. Because the breakdown of long gaps occurs mostly when the Tesla voltage is negative, we concentrated on the discharges that took place around two significant negative peaks (t bd = 26.4 and 36.1 µs). Data scattering is not significant. Considering again the breakdown events at high U bd and I max (red group in Figure 7(c)), we can estimate R = 1.7 k. If we study the discharges occurring at lower voltage (around 230 kV), then we find an operational resistance R = 3.3 k.

Measuring the guided discharge characteristics for each value of L enables us to plot the evolution of the operational resistance R with L (see Figure 8). To this purpose, we used the aforementioned method and only selected the data in the breakdown voltage range -360 kV < U bd < -300 kV. Fitting this curve by a linear function gives a lineic resistance of 16 /cm. FIG. 8. Evolution of the operational resistance R with the gap length L (scatter plot) and linear fit (red solid line). Only breakdown occurring in the voltage range -360 kV < U bd < -300 kV were taken into account.

IV. DISCUSSION

The discharge dynamics can be described by a simple modeling. Since the current flows for about 100 ns in the plasma, its origin lies in the discharge of the Tesla output capacitor C 2 , and not in the voltage oscillations of the Tesla coil, of which period is far larger (T ~10 µs). We can thus write:

(

) 1
where Q is the charge in the equivalent capacitor C out and  corresponds to the full width at half maximum (FWHM) of the main current peak. A systematic investigation of the observed discharges for all gaps gives, for the Tesla coil output capacitor, a value , which is lower than the measured value for C 2 (49 pF). We explain this discrepancy by leakages of the initial charge to the ground and through coronas. Indeed, partial corona discharges were already observed in the case of laser-guided discharges, as seen in [26]. More recently, streak images showing the development of a laser-guided discharge using a Tesla coil exhibited transient ionized channels appearing and disappearing with the generator oscillating voltage prior to the main discharge [23]. The feeding of charges in such transitory channels could also explain the charge loss observed in our experiments. Using this crude modeling, we can therefore estimate the discharge resistance as:

(2) which, for (corresponding to a gap length L = 50 cm) gives R = 900 , close to the measured operational resistance. Identifying the Tesla output voltage with the output capacitor voltage, we can also write its time evolution as:

(3) being the total discharge current, including the leakages. Assuming that the measured current I and the total current are related by one is then able to evaluate the gap voltage temporal evolution from the current measurements. However, estimating the plasma resistance at the time when the current is maximal is easier. Since the main current peak is approximately symmetric, this gives immediately:

(4)

Using this formula the guided discharge lineic resistance for the set of data in the breakdown voltage range -360 kV < U bd < -300 kV (see Figure 8) can then be estimated to 8 /cm. The use of an AC generator to produce laser guided discharges leads to very specific experimental conditions when compared to the frequently used monopolar pulsed or DC sources. The dynamics for the discharge development is thought to be quite different in the case of an oscillating electric field. Previous study has proved that for centimeter-scale gaps in case of DC discharges, the initial plasma generated by filamentation has a too short lifetime (about 1 ns) to cause breakdown by itself [27]. Instead, this plasma column undergoes a significant Joule heating in the presence of external electric field, causing the hydrodynamic expansion of the filament and in turn lowering the breakdown voltage in the resulting underdense gas channel. If we assume this scenario is also valid for longer gaps, we can explain the evolution of the breakdown probability with t laser (see Figure 4). Indeed, if filamentation occurs when the gap electric field is strong, the resulting heating effect will be important, leading to the formation of a low-density channel. On the contrary, if the filamentation plasma is formed in a very weak external field, the heating effect will be almost inexistent. Still, even if it is low, breakdown probability in this case is not exactly zero. We even managed to score discharges when the laser was sent up to a few microseconds before the Tesla triggering, and this effect was also witnessed by other groups [19,28,16]. This could be explained by the fact that even in absence of external field, filamentation result in a low-density air channel over a microsecond timescale, as recently observed by Cheng et al. [29]. Thus, the external field only speeds up and enhances this hydrodynamic effect.

As for the discharge development taking place between t laser and t bd , it has been shown to be dictated by the Tesla voltage oscillations [23]. In this paper, streak images displayed the progression of an ionized leader in the gap, which disappeared each time the Tesla voltage turned to 0, and reappearing when it reached its local extremum. This behavior can explain why long gap breakdown can only be achieved by sending the laser at an early time. By doing so, the gap is exposed during a long time to the oscillating voltage, of which amplitude is rising. If the laser is sent later, the voltage has already passed the first half of the beating period, which means that the amplitude of the voltage peaks will be decreasing, effectively inhibiting the breakdown development.

V. SUMMARY

Laser guided discharges induced by laser filamentation in an oscillating electric field from a compact Tesla coil have been investigated. These voltage oscillations are characterized by a 9.6 µs period and maximum amplitude of 365 kV. Studies concentrated on the first half of the initial voltage beating period, characterized by an increasing amplitude for voltage extrema. The maximum gap length for self-breakdown has been measured to L max = 36 cm. With the laser filaments, we were able to achieve a 50 % breakdown probability for L = 140 cm, and scored discharges for gaps up to L = 180 cm, corresponding to an increase of the discharge length by a factor 5. With a 1 m gap, 10 Hz repetitive guided discharges were also demonstrated.

Sending the laser when the Tesla voltage was maximum created the most favorable conditions for discharges, with a noticeable dependence on the voltage polarity. Thus, more breakdowns were reported when the electric field direction was opposite to the laser propagation direction at the time of filament creation.

The onset of a guided discharge is also strongly correlated to the Tesla voltage local maxima, as breakdowns occur preferentially near such extrema. It is interesting to note that the delay between the laser shot and the breakdown is quite large, on the order of the period of the voltage oscillations (T = 9.6 µs). Long gaps require a longer time to close than short gaps, and can require up to 2T before leading to breakdown.

Discharge current consists in strongly damped oscillations, with a main peak of which shape is close to a Gaussian curve. The surface of this peak remains approximately constant for different gaps, with short gaps resulting in a higher and narrower shape. Thus, a gap L = 40 cm gives a current pulse of about 350 A for a 35 ns width while L = 100 cm results in a 200 A, 60 ns peak. We were also able to estimate the plasma resistance at peak current. The evolution of this value with L is close to linear, yielding a lineic resistance of 8 /cm.

FIG. 1 .

 1 FIG. 1. (a) Photograph of the Tesla coil used in the experiments. (b) Equivalent electric schema.

Figure 2 (

 2 b)). The occurrence of breakdown is also clearly visible on the antenna signal in the shape of exponentially damped high-frequency oscillations (Figure 2(b)).

FIG. 2 .

 2 FIG. 2. (a) Antenna signal generated by the Tesla coil in absence of discharge. (b) Antenna signal in the presence of a laser induced discharge.

FIG. 4 .

 4 FIG. 4. Evolution of the breakdown probability P with t laser . (a): L = 50 cm, configuration 1 (red solid curve and squares) and configuration 2 (green solid curve and circles). (b) L = 100 cm, configuration 1 (red solid curve and squares) and L = 90 cm, configuration 2 (green solid curve and circles).

FIG. 5 .

 5 FIG. 5. t as a function of t laser for L = 50 cm (a) and L = 100 cm (b) (scatter plots). The oblique lines represent the time position of positive (dotted lines) and negative (dashed lines) Tesla voltage peaks. The evolution of the Tesla voltage (blue solid line) in time is also given for convenience.

FIG. 6 .

 6 FIG. 6. Typical discharge current curves for L = 50 cm (red solid line) and L = 100 cm (blue dashed line).

FIG 7 .

 7 FIG 7. (I max , U bd ) characteristics for L = 40 and 50 cm. (a): positive quadrant. (b): negative quadrant. (c) Negative quadrant of the (I max , U bd ) characteristic for L = 100 cm.

Y.-H. Cheng, J. K. Wahlstrand, N. Jhajj and H. M. Milchberg, Opt. Express 21, 4740 (2013).

Acknowledgements

This work was supported by the French Direction Générale de l'Armement (grant n° 2007 95 091). The authors would like to thank Dr. Y. Liu, A. Dos Santos and Jean-Lou Charles for technical support.


Guillaume Bouguen

Laurent Dubuquoy

Pierre Desreumaux

Thomas Brunner

PhD Benjamin Bertin
email: benjamin.bertin-2@univ-lille2.fr

Intestinal steroidogenesis

Keywords: Intestinal steroidogenesis, glucocorticoids, intestinal epithelial cells, Liver receptor homolog-1, inflammatory bowel diseases, colorectal cancer

Steroids are fundamental hormones that control a wide variety of physiological processes such as metabolism, immune functions, and sexual characteristics. Historically, steroid synthesis was considered a function restricted to the adrenals and the gonads. In the past 20 years, a significant number of studies have demonstrated that steroids could also be synthesized or metabolized by other organs. According to these studies, the intestine appears to be a major source of de novo produced glucocorticoids as well as a tissue capable of producing and metabolizing sex steroids. This finding is based on the detection of steroidogenic enzyme expression as well as the presence of bioactive steroids in both the rodent and human gut. Within the intestinal mucosa, the intestinal epithelial cell layer is one of the main cellular sources of steroids. Glucocorticoid synthesis regulation in the intestinal epithelial cells is unique in that it does not involve the classical positive regulator steroidogenic factor-1 (SF-1) but a closely related homolog, namely the liver receptor homolog-1 (LRH-1). This local production of immunoregulatory glucocorticoids contributes to intestinal homeostasis and has been linked to pathophysiology of inflammatory bowel diseases. Intestinal epithelial cells also possess the ability to metabolize sex steroids, notably estrogen; this mechanism may impact colorectal cancer development. In this review, we contextualize and discuss what is known about intestinal steroidogenesis and regulation as well as the key role these functions play both in physiological and pathological conditions.

Introduction

In vertebrates, the mucosal surfaces provide a barrier against invading pathogenic microorganisms but represent also an essential interface exchange between the host and its environment [START_REF] Sansonetti | War and peace at mucosal surfaces[END_REF]. With a surface of broadly 300m 2 in adults, the intestinal mucosa is the biggest mucosal surface in humans. The gastrointestinal tract is constantly colonized by a huge number of microorganisms (approximately 100 trillion), mostly bacteria, but also viruses and fungi, which are collectively referred to as the microbiota [START_REF] Walter | The human gut microbiome: ecology and recent evolutionary changes[END_REF]. This colonization leads to a complex mutualistic relationship between the host and the microbiota in which each partner contributes positively to various physiological processes of the other one. For example, the microbiota digests and ferments carbohydrates that provide nutrients to the host cell, produces essential vitamins, contributes to the development of the gut immune system, and protects the host from potential pathogens. In turn, the host regulates microbial ecology by providing nutrients and ecological niches. It also controls microbial density trough the synthesis of antimicrobial peptides and immunoglobulins. This dialog is required to maintain what is commonly called intestinal homeostasis. The disruption of this homeostasis may lead to the development of pathologies such as inflammatory bowel diseases [START_REF] Kamada | Role of the gut microbiota in immunity and inflammatory disease[END_REF][START_REF] Maloy | Intestinal homeostasis and its breakdown in inflammatory bowel disease[END_REF].

A fundamental actor in the maintenance of the gut immune homeostasis are the intestinal epithelial cells (IECs) [START_REF] Peterson | Intestinal epithelial cells: regulators of barrier function and immune homeostasis[END_REF][START_REF] Goto | Intestinal epithelial cells as mediators of the commensal-host immune crosstalk[END_REF]. IECs, located in the intestinal mucosa, provide a physical and biochemical barrier that separates microbiota and immune cells. In addition, interactions of IECs with the microbiota or with pathogens result in anti-microbial and immunoregulatory responses trough the production of numerous molecules [START_REF] Peterson | Intestinal epithelial cells: regulators of barrier function and immune homeostasis[END_REF][START_REF] Rimoldi | Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells[END_REF][START_REF] Iliev | Intestinal epithelial cells promote colitis-protective regulatory T-cell differentiation through dendritic cell conditioning[END_REF][START_REF] Iliev | Human intestinal epithelial cells promote the differentiation of tolerogenic dendritic cells[END_REF]. One immunoregulatory molecule produced locally by IECs is represented by glucocorticoids (GCs; i.e. cortisol in humans and corticosterone in rodents).

Historically, corticosteroid (glucocorticoids and mineralocorticoids) synthesis only occurred in the adrenal cortex. A significant number of studies have now challenged this view by demonstrating that organs such as the thymus [START_REF] Vacchio | Steroid production in the thymus: implications for thymocyte selection[END_REF][START_REF] Pazirandeh | Paracrine glucocorticoid activity produced by mouse thymic epithelial cells[END_REF][START_REF] Lechner | Glucocorticoid production in the murine thymus[END_REF], the skin [START_REF] Thiboutot | Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1)[END_REF], the brain [START_REF] Gomez-Sanchez | Corticosteroid synthesis in the central nervous system[END_REF][START_REF] Croft | Effects of minor laboratory procedures, adrenalectomy, social defeat or acute alcohol on regional brain concentrations of corticosterone[END_REF][START_REF] Little | Selective increases in regional brain glucocorticoid: a novel effect of chronic alcohol[END_REF], the intestine [START_REF] Cima | Intestinal epithelial cells synthesize glucocorticoids and regulate T cell activation[END_REF][START_REF] Sidler | Colon cancer cells produce immunoregulatory glucocorticoids[END_REF][START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF], and the lung [START_REF] Hostettler | Local glucocorticoid production in the mouse lung is induced by immune cell stimulation[END_REF] are capable of producing gluco-or mineralocorticoids. This review aims to present evidence of local steroid synthesis, particularly that of GCs in the intestine, as well as the key factors involved in the regulation of this production. We discuss the known and potential roles of GCs in the regulation of intestinal homeostasis and then review the data showing that IECs, aside from their ability to produce GCs, are also involved in the metabolism of other steroids (notably sex steroids).

Intestinal Glucocorticoids

Evidence of intestinal GC production

The steroidogenic ability of the gut was first suggested in the beginning of the 90's after looking for the expression and activity of the CYP17 (P450c17) enzyme along the gastrointestinal tract of adult rats (see Figure 1 for a schematic representation of the cortisol and sex steroids synthesis pathway). When Dalla Valle et al. assessed the ex vivo metabolism of [4-14C]pregnenolone in various organs, they found that the duodenum and the colon were able to produce high amounts of dehydroepiandrosterone (DHEA), attesting the presence of 17-hydroxylase and C 17,20-lyase activities in the intestine [START_REF] Valle | Extraglandular hormonal steroidogenesis in aged rats[END_REF][START_REF] Valle | Occurrence of cytochrome P450c17 mRNA and dehydroepiandrosterone biosynthesis in the rat gastrointestinal tract[END_REF]. While rodents are corticosterone-secreting animals, due to the lack of expression of CYP17 in the adrenal, CYP17 expression in peripheral tissues (notably in the intestine) could lead to the synthesis of local cortisol in rats or mice. However, an early study failed to identify GCs (corticosterone or cortisol) in the small intestine of rats by thin layer chromatography [START_REF] Vianello | Developmentally regulated expression and activity of 17alpha-hydroxylase/C-17,20-lyase cytochrome P450 in rat liver[END_REF]. Moreover, the hypothesis of locally produced cortisol in rodents has never been tested and only corticosterone has been investigated.

In the same period of time, the identification of the transcripts encoding both CYP11A1 (cholesterol side-chain cleavage cytochrome P450; P450scc) and 3HSD (3 beta-hydroxysteroid dehydrogenase) in the primitive gut of the mouse embryo suggested the ability of the gut to synthesize steroids de novo [START_REF] Keeney | Cholesterol side-chain cleavage cytochrome P450 gene expression in the primitive gut of the mouse embryo does not require steroidogenic factor 1[END_REF]. The expression of 11HSD2 (11 betahydroxysteroid dehydrogenase 2), which converts active GCs to inactive metabolites, was also detected in the intestine of rodents and humans [START_REF] Pacha | Distribution of 11 beta-hydroxysteroid dehydrogenase along the rat intestine[END_REF][START_REF] Whorwood | Epithelial cell localization of type 2 11 betahydroxysteroid dehydrogenase in rat and human colon[END_REF]. Subsequent studies demonstrated steroidogenic enzyme expression as well as intestinal steroid metabolism in various species such as frog [START_REF] Belvedere | Hormonal steroidogenesis in liver and small intestine of the green frog, Rana esculenta L[END_REF], fish [START_REF] Wang | Cloning of zebrafish ovarian P450c17 (CYP17, 17alpha-hydroxylase/17, 20-lyase) and characterization of its expression in gonadal and extra-gonadal tissues[END_REF], mouse [START_REF] Cima | Intestinal epithelial cells synthesize glucocorticoids and regulate T cell activation[END_REF], and human [START_REF] Sidler | Colon cancer cells produce immunoregulatory glucocorticoids[END_REF][START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF][START_REF] Coste | LRH-1mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease[END_REF]. Most of the comprehensive studies on intestinal steroidogenesis have been undertaken using mouse models. Cima et al. first measured (by radioimmunoassay) the de novo synthesis of corticosterone in murine small intestinal tissue fragments cultured ex vivo [START_REF] Cima | Intestinal epithelial cells synthesize glucocorticoids and regulate T cell activation[END_REF]. This production was highly stimulated in response to immune activation of T lymphocytes.

Corticosterone synthesis was proven to be in situ, since GC release from tissue was blocked by metyrapone, a11 hydroxylase (P450c11; CYP11B1) inhibitor, and 11HSD1 [START_REF] Cima | Intestinal epithelial cells synthesize glucocorticoids and regulate T cell activation[END_REF][START_REF] Dominguez | Mechanism of Inhibition of Adrenal Steroid 11beta-Hydroxylase by Methopyrapone (Metopirone)[END_REF][START_REF] Sampath-Kumar | Metyrapone is a competitive inhibitor of 11beta-hydroxysteroid dehydrogenase type 1 reductase[END_REF]. CYP11A1, 3HSD, CYP21, CYP11B1, and 11HSD1 enzymes were all expressed in the murine small and large intestinal mucosa, but some of them were only detected after immune stimulation [START_REF] Cima | Intestinal epithelial cells synthesize glucocorticoids and regulate T cell activation[END_REF]. The cellular source of steroidogenic enzymes and GCs was shown to be the IECs located in the crypt region of the small intestine [START_REF] Cima | Intestinal epithelial cells synthesize glucocorticoids and regulate T cell activation[END_REF]. Further study demonstrated that the expression patterns of mRNA encoding CYP11A1 and CP11B1 were linked to cell cycle. This restricts the main intestinal GC source to the proliferative cells of the intestinal crypts [START_REF] Atanasov | Cell cycle-dependent regulation of extra-adrenal glucocorticoid synthesis in murine intestinal epithelial cells[END_REF]. Cima et al. suggested that GC synthesis could also occur in the human intestine, although results were not shown [START_REF] Cima | Intestinal epithelial cells synthesize glucocorticoids and regulate T cell activation[END_REF]. Earlier observations suggested intestinal steroid metabolism in humans following the detection of 11HSD and 17 beta-hydroxysteroid dehydrogenase (17HSD) enzymes in the colon and along the gastrointestinal tract [START_REF] Whorwood | Epithelial cell localization of type 2 11 betahydroxysteroid dehydrogenase in rat and human colon[END_REF][START_REF] Sano | 17 beta-Hydroxysteroid dehydrogenase type 2 expression and enzyme activity in the human gastrointestinal tract[END_REF].

Initial work by

More recently, CYP11A1, CYP17 and CYP11B1 and a production of cortisol were observed in colorectal cancer cell lines (Caco-2, HT-29) as well as in primary human intestinal tissue and colorectal tumors [START_REF] Sidler | Colon cancer cells produce immunoregulatory glucocorticoids[END_REF]. Caco-2 cells were also found to constitutionally express 3HSD1 and 2 [START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF], as well as the cholesterol transporter StAR, CYP21 and 11HSD1 (B. Bertin, unpublished data). In human tissue, CYP11A1 and CYP11B1 were detected in colonic biopsies suggesting that in physiological state the human mucosa is able to produce GC [START_REF] Coste | LRH-1mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease[END_REF].

We recently confirmed and detailed this finding by showing expression of steroidogenic enzymes (3HSD 1, 3HSD 2, 11HSD1) and cortisol production in colonic epithelial cells isolated from human surgical specimens [START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF].

Regulation of GC production: from adrenal to the intestine

In the adrenal, the regulation of cortisol production is well characterized and is the result of a complex interaction between three endocrine glands (hypothalamus, pituitary, and adrenal; HPA), which constitute the HPA axis [START_REF] Papadimitriou | Regulation of the hypothalamic-pituitary-adrenal axis[END_REF]. Hypothalamic hormones (corticotropin-releasing hormone and vasopressin) induce peptidic adrenocorticotropic hormone (ACTH) secretion in the anterior pituitary, which regulates the synthesis and release of cortisol from the adrenal cortex [START_REF] Papadimitriou | Regulation of the hypothalamic-pituitary-adrenal axis[END_REF]. ACTH acts on the adrenal steroidogenesis through the stimulation of cholesterol transporter expression and that of the steroidogenic enzymes [START_REF] Sewer | Insights into the transcriptional regulation of steroidogenic enzymes and StAR[END_REF]. The protein SF-1 (steroidogenic factor-1) is one of the key regulators in the expression of these steroidogenic factors in the adrenals. SF-1 belongs to the superfamily of nuclear receptors, which are mainly ligand-dependent transcription factors. SF-1 binds to the promoter and induces the expression of StAR, CYP 450 steroid hydroxylases, -hydroxysteroid dehydrogenases as well as melanocortine-2 receptor (MC2R), the receptor for ACTH (reviewed in [START_REF] Schimmer | Minireview: steroidogenic factor 1: its roles in differentiation, development, and disease[END_REF]). The phenotype of adrenocortical (and gonadal) insufficiency in SF-1 knockout mice clearly established this transcription factor as pivotal in the development and physiology of steroidogenic tissues [START_REF] Luo | A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation[END_REF][START_REF] Parker | Steroidogenic factor 1: a key determinant of endocrine development and function[END_REF]. However, SF-1 is not expressed in the intestinal mucosa [START_REF] Ramayya | Steroidogenic factor 1 messenger ribonucleic acid expression in steroidogenic and nonsteroidogenic human tissues: Northern blot and in situ hybridization studies[END_REF][START_REF] Mueller | Differential regulation of glucocorticoid synthesis in murine intestinal epithelial versus adrenocortical cell lines[END_REF]. SF-1 belongs to the nuclear receptor subfamily 5, group A, along with its close homolog LRH-1 (liver receptor homolog-1), which is highly expressed in intestinal crypt cells [START_REF] Botrugno | Synergy between LRH-1 and beta-catenin induces G1 cyclin-mediated cell proliferation[END_REF], suggesting LRH-1's potential role in the regulation of intestinal steroidogenesis. Indeed, this was first demonstrated in vitro by Mueller and colleagues who showed that LRH-1 induces the CYP11A1 and CYP11B1 expression as well as corticosterone production in the murine intestinal epithelial cell line [START_REF] Mueller | Differential regulation of glucocorticoid synthesis in murine intestinal epithelial versus adrenocortical cell lines[END_REF][START_REF] Mueller | The nuclear receptor LRH-1 critically regulates extra-adrenal glucocorticoid synthesis in the intestine[END_REF]. In human colon cancer cell lines, the down regulation of LRH-1 by small interfering RNA leads to the reduction of steroidogenic enzyme expression and cortisol synthesis [START_REF] Sidler | Colon cancer cells produce immunoregulatory glucocorticoids[END_REF][START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF]. In normal human colonic epithelial cells, the expression level of the mRNA encoding LRH-1 strongly correlates with the expression levels of 3HSD and 11HSD [START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF], suggesting that LRH-1 regulates intestinal GC synthesis under physiological state. In vivo, LRH-1 haploinsufficiency in mice confirmed the critical role of LRH-1 in controlling intestinal corticosterone production. Upon activation of T lymphocytes, CYP11A1 and CYP11B1 mRNA levels and corticosterone release were reduced in the intestine of heterozygous LRH-1 +/-mice [START_REF] Mueller | The nuclear receptor LRH-1 critically regulates extra-adrenal glucocorticoid synthesis in the intestine[END_REF][START_REF] Noti | Lipopolysaccharide induces intestinal glucocorticoid synthesis in a TNFalpha-dependent manner[END_REF].

The differential regulation of glucocorticoid synthesis in adrenal and intestinal cells is also illustrated by the observation that ACTH administration was able to trigger the release of corticosterone from the adrenal but not from the intestine [START_REF] Mueller | Differential regulation of glucocorticoid synthesis in murine intestinal epithelial versus adrenocortical cell lines[END_REF]. ACTH mainly mediates its effect through the stimulation of adenylate cyclase leading to an increase of intracellular cyclic adenosine monophosphate (cAMP) level and subsequent protein kinase A activation. Thus, it was observed that increasing intracellular cAMP level in the adrenocortical tumor cell line Y1 induces the expression of CYP11A1 and CYP11B1, while the opposite effect was obtained in the murine intestinal epithelial cell line mIC c12 [START_REF] Mueller | Differential regulation of glucocorticoid synthesis in murine intestinal epithelial versus adrenocortical cell lines[END_REF]. Conversely, it was shown that phorbolester-dependent protein kinase C activation was involved in the control of the steroidogenic enzyme expression and GC production in mIC c12 cells but not in Y1 cells [START_REF] Mueller | Differential regulation of glucocorticoid synthesis in murine intestinal epithelial versus adrenocortical cell lines[END_REF].

However, LRH-1 mRNA level was slightly modified by cAMP and phorbolester in mIC c12 .

Recently Lee and colleagues observed in Hela cell lines the ability of phorbolester to stimulate the phosphorylation of LRH-1 in an ERK1/2-dependent manner [START_REF] Lee | Phosphorylation of the hinge domain of the nuclear hormone receptor LRH-1 stimulates transactivation[END_REF]. Therefore, rather than a modification of LRH-1 expression, different phosphorylation states of LRH-1 may explain the antagonistic effect of cAMP and phorbolester observed in intestinal epithelial cells [START_REF] Mueller | Differential regulation of glucocorticoid synthesis in murine intestinal epithelial versus adrenocortical cell lines[END_REF]. The underlying reasons of the discrepancy between adrenal and intestinal cells during the regulation of cortisol synthesis remain currently unknown and the control of intestinal steroidogenesis by one or several hormonal signals needs to be characterized.

To date, only the induction of strong immune responses has been found to induce intestinal steroidogenesis. Pioneering works have demonstrated that de novo synthesis of corticosterone from small intestinal tissue fragments was highly stimulated in response to immune activation of T lymphocytes [START_REF] Cima | Intestinal epithelial cells synthesize glucocorticoids and regulate T cell activation[END_REF]. Using lipopolysaccharide (LPS) injection in mice, subsequent studies investigated whether activation of the innate immune system could also induce intestinal steroidogenesis. LPS was able to induce intestinal CYP11A and CYP11B1 expression level and intestinal corticosterone release in RAG2 -/-mice (which lack T and B lymphocytes), suggesting that intestinal GC synthesis induction also occurs independently of the activation of the adaptive immune system [START_REF] Noti | Lipopolysaccharide induces intestinal glucocorticoid synthesis in a TNFalpha-dependent manner[END_REF]. The authors showed that the cells, which contribute to this LPS-induced intestinal steroidogenesis, were macrophages [START_REF] Noti | Lipopolysaccharide induces intestinal glucocorticoid synthesis in a TNFalpha-dependent manner[END_REF].

Finally, the pro-inflammatory cytokine tumor necrosis factor (TNF) was clearly demonstrated to be the common mediator that regulates intestinal GC production in response to the activation of both the innate and adaptative immune system [START_REF] Noti | Lipopolysaccharide induces intestinal glucocorticoid synthesis in a TNFalpha-dependent manner[END_REF]. Following immune stimulation by LPS or activation of T cells, intestinal GC synthesis was compromised both in mice lacking TNF cytokine or TNF receptor expression. The involvement of TNF was later confirmed but the precise role of this cytokine needs further clarifications since opposite effects on gut steroidogenesis were found [START_REF] Noti | TNF suppresses acute intestinal inflammation by inducing local glucocorticoid synthesis[END_REF][START_REF] Huang | Tumor necrosis factor suppresses NR5A2 activity and intestinal glucocorticoid synthesis to sustain chronic colitis[END_REF]. One might argue that the differential involvement of LRH-1 according to the immune response may explain such discrepancies. In wild type mice, both activated T cells and LPS were able to induce CYP11B1 expression and corticosterone production in a TNF-dependent manner, but only the T cell-dependent induction of intestinal steroidogenesis was compromized in LRH-1 haplodeficient animals (LRH-1 +/-), which suggest that LPS drives intestinal GC production independently of LRH-1 [START_REF] Noti | Lipopolysaccharide induces intestinal glucocorticoid synthesis in a TNFalpha-dependent manner[END_REF]. By contrast, Huang et al suggested that TNF-dependent pro-inflammatory signals mediated by NF-kB and JNK suppressed CYP11A1 and CYP11B1 expression by inhibiting LRH-1 transcriptional activity under chronic inflammatory conditions [START_REF] Huang | Tumor necrosis factor suppresses NR5A2 activity and intestinal glucocorticoid synthesis to sustain chronic colitis[END_REF]. In summary, the understanding of the regulatory mechanisms of GC synthesis in the gut is just at the beginning, and additional mechanisms involved in the regulation of intestinal steroidogenesis remain to be discovered.

Function and roles of intestinal GCs

Maturation and maintenance of the intestinal epithelial barrier.

GC-mediated effects on the maturation of the small intestine are well recognized both in rodents and humans [START_REF] Drozdowski | Intestinal hormones and growth factors: effects on the small intestine[END_REF]. For instance, adrenalectomy in rats during the weaning period impairs the morphological development of the small intestine [START_REF] Miyata | Impaired growth of small intestinal epithelium by adrenalectomy in weaning rats[END_REF], while injection of cortisone or hydrocortisone to suckling rats increases the expression and activity of many small intestinal maturation markers [START_REF] Yeh | Thyroxine and cortisone cooperate to modulate postnatal intestinal enzyme differentiation in the rat[END_REF][START_REF] Schaeffer | Cytokine gene expression during postnatal small intestinal development: regulation by glucocorticoids[END_REF][51] and paracellular permeability, they showed that synthetic GCs (prednisolone and dexamethasone) had a protective effect against the TNF-dependent increase of intestinal permeability [START_REF] Boivin | Mechanism of glucocorticoid regulation of the intestinal tight junction barrier[END_REF]. In vitro, a positive effect of dexamethasone on epithelial barrier function was subsequently found independent of the presence of any immunological stimulus [START_REF] Fischer | Glucocorticoids regulate barrier function and claudin expression in intestinal epithelial cells via MKP-1[END_REF].The overall in vitro response of human intestinal epithelial cells to GCs was also studied using microarray analysis. This work showed that hydrocortisone differentially regulates the expression levels of protein encoding genes and enterocyte markers involved in polarization and tight junction formation [START_REF] Lu | Hydrocortisone induces changes in gene expression and differentiation in immature human enterocytes[END_REF]. Altogether, these studies underlined that both endogenous (adrenal-derived) and exogenous GCs play a role during the functional maturation of the gut and the maintenance of intestinal epithelial barrier function. However, during these processes the effective contributions of GCs produced locally by the intestine remain to be established.

As compared to the adrenal, the amount of GCs synthesized by IECs under steady state conditions is rather low, and to date there is no experimental evidence of the impact of intestinal GCs on intestinal barrier maturation and/or maintenance. It was previously reported that the specific deletion of LRH-1 in mouse IECs disrupted intestinal steroidogenesis and rendered the conditional knockout animals more susceptible to experimental colitis ( [START_REF] Coste | LRH-1mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease[END_REF], see paragraph 4.1). Although a change of intestinal permeability due to the LRH-1-dependent decrease of intestinal GC could explain this phenotype, this hypothesis remains to be tested.

Using a short hairpin RNA strategy, we recently created a stable Caco-2 cell line in which the expression of LRH-1 was reduced by 80% compared to a control cell line [START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF]. This downregulation of LRH-1 resulted in a reduction of cortisol synthesis and release. However, preliminary experiments did not observed any change of the TEER of this stable cell line up to 21 days when it was differentiated and grown as a monolayer on filters, suggesting that intestinal-derived cortisol may not participate in the development of intestinal barrier (author's unpublished data). Further experiments are needed to investigate the potential role of intestinal GCs on gut epithelial differentiation and barrier function, e.g. the measurement of intestinal permeability of LRH-1-deficient mice under steady-state and inflammatory conditions.

Impact on innate and adaptative immune responses.

Consistent with the general immunoregulatory properties of GC [START_REF] Rhen | Antiinflammatory action of glucocorticoids--new mechanisms for old drugs[END_REF][START_REF] Bosscher | Minireview: latest perspectives on antiinflammatory actions of glucocorticoids[END_REF], as well as the induction of intestinal steroidogenesis following immune activation, it is likely that local GC release is involved in the regulation of intestinal immune responses. In order to specifically appreciate the role of GCs released by extra-adrenal sources (such as the intestine), Cima and colleagues removed adrenal glands, and challenged these mice with various T cell-dependent immunological stresses [START_REF] Cima | Intestinal epithelial cells synthesize glucocorticoids and regulate T cell activation[END_REF]. Adrenalectomized animals were notably infected with lymphocytic choriomeningitis virus (LCMV) (which triggers a robust cytotoxic T lymphocyte response) in the presence or absence of the GC synthesis inhibitor metyrapone. Then, the virus-specific T cell activation in the intestine was assessed by monitoring membrane markers expression and cytokine production. LCMV-infected and metyrapone-treated adrenalectomized mice displayed a higher T cell activation compared to LCMV-infected animals only, indicating that de novo synthesized extra-adrenal corticosterone inhibited the T cell response. Although this work did not formally demonstrate the intestine as the only source of GCs, results obtained by Cima et al. strongly suggested its impact. [START_REF] Mueller | The nuclear receptor LRH-1 critically regulates extra-adrenal glucocorticoid synthesis in the intestine[END_REF]. Regarding specifically the gut, heterozygous LRH-1 +/-mice, which present a decreased expression of steroidogenic enzymes and corticosterone release were more susceptible to experimental colitis induced by either 2,4,6-trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulfate (DSS) [START_REF] Coste | LRH-1mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease[END_REF]. However LRH-1 expression was reduced in all cell types of rodents, making it difficult to exclude the contribution of GCs produced by other cells, such as immune cells [START_REF] Jia | Steroidogenic enzyme Cyp11a1 regulates Type 2 CD8+ T cell skewing in allergic lung disease[END_REF]. But the immunoregulatory role of GCs synthesized by the IECs was further supported by more specific experiments in the same study when enterocyte-specific LRH-1-deficient mice produced less colonic corticosterone after TNBS treatment and displayed more severe inflammatory lesions in the large intestine [START_REF] Coste | LRH-1mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease[END_REF] Beside the immunoregulatory properties of GCs, other mechanisms such as the maintenance of the intestinal barrier and/or the recovery of the epithelial layer by promoting progenitor cell proliferation through LRH-1 might also regulate colitis. However, chemically-induced models of colitis imperfectly reproduced the cellular and molecular aspects of the physiopathology of inflammatory bowel diseases (IBD), these results led also to the assessment of the expression of LRH-1 and intestinal GCs synthesis in patients suffering from IBD (see paragraph 4.1).

Apart from the characterization of T lymphocytes as targets of intestinal GCs, no other cellular target has been described yet. It has recently been demonstrated that a fundamental dialog between IEC and dendritic cells (DCs) takes place within the intestinal mucosa to maintain gut homeostasis. DCs are professional antigen presenting cells (APC) viewed as one of the gate keepers of the immune system forming a bridge between the innate and adaptive immune responses. Recent studies demonstrated that IECs educate intestinal DCs to suppress inflammation and promote tolerance and gut immune homeostasis [START_REF] Rimoldi | Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells[END_REF][START_REF] Iliev | Human intestinal epithelial cells promote the differentiation of tolerogenic dendritic cells[END_REF][START_REF] Zeuthen | Epithelial cells prime the immune response to an array of gut-derived commensals towards a tolerogenic phenotype through distinct actions of thymic stromal lymphopoietin and transforming growth factor-beta[END_REF]. Several immunoregulatory molecules, such as TGF- (transforming growth factor-), retinoic-acid or TSLP (thymic stromal lymphopoietin), produced by IECs are crucially involved in this tolerogenic process [START_REF] Peterson | Intestinal epithelial cells: regulators of barrier function and immune homeostasis[END_REF]. Therefore, it is tempting to speculate that immunoregulatory GCs released by IECs might have a similar role in promoting the development of DCs (or others APC) with tolerogenic properties. All the same, this hypothesis remains to be tested.

Other properties of intestinal GCs

Next to GR-mediated effects regulation of GC-sensitive genes, there is also accumulating evidence for non-genomic effects of GCs. For example, GCs have been reported to mediate certain biological effects via interaction with cellular membranes, via cytosolic GR and via membrane-bound GC receptors [START_REF] Stahn | Molecular mechanisms of glucocorticoid action and selective glucocorticoid receptor agonists[END_REF]. Some gut-specific properties have been allocated to this intestinal steroidogenesis. Cortisol released by IECs has been recently demonstrated to be involved in the control of the intestinal expression of PPAR (peroxisome proliferator activated receptor gamma), a key factor in the regulation of gut homeostasis. The colon is a major PPAR-expressing tissue with the colonic epithelial cells as the main source of expression [START_REF] Fajas | The organization, promoter analysis, and expression of the human PPARgamma gene[END_REF][START_REF] Dubuquoy | PPARgamma as a new therapeutic target in inflammatory bowel diseases[END_REF]. PPAR belongs to the nuclear receptor superfamily and has been shown to be a critical regulator of inflammatory responses [START_REF] Dubuquoy | PPARgamma as a new therapeutic target in inflammatory bowel diseases[END_REF][START_REF] Ricote | The peroxisome proliferatoractivated receptor-gamma is a negative regulator of macrophage activation[END_REF][START_REF] Jiang | PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines[END_REF][START_REF] Tontonoz | Fat and beyond: the diverse biology of PPARgamma[END_REF][START_REF] Glass | Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells[END_REF]. This property involves typical positive gene regulation by binding of the PPAR -RXR (RXR, retinoid X receptor) heterodimer to the PPAR-response element (PPRE) located in the promoter of target genes. In addition a direct interference with transcription factors involved in the inflammatory response, such as NF-B (nuclear factor kappa-light-chainenhancer of activated B cells), AP-1 (activating protein-1) and ROR t (retinoic acid receptorrelated orphan receptor gamma t), has been demonstrated in a mechanism often referred to as "transrepression" [START_REF] Glass | Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells[END_REF]. In a stable Caco2-cell line with effective LRH-1 knockdown cortisol production and release, as well as PPAR mRNA and protein expression were drastically reduced [START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF]. This inhibition of PPAR expression was reversed to the normal levels following the treatment of the cell line by exogenous GCs. As a control, knockdown of PPAR by the same strategy did not result in a decrease of cortisol release or LRH-1 expression [START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF]. These experiments pointed out a key role of intestinal cortisol synthesis in the regulation of intestinal PPAR and PPAR -mediated gut homeostasis.

Environmental stresses induce adaptive response of IECs with high secretory activity. The endoplasmic reticulum (ER) folds and matures most secreted and transmembrane proteins.

The imbalance between protein load observed in stress situation and folding ability of the ER is defined as ER stress, and the coordinated response triggered is known as UPR (Unfold Protein Response) to ensure homeostatic cell control [START_REF] Ron | Signal integration in the endoplasmic reticulum unfolded protein response[END_REF][START_REF] Todd | The endoplasmic reticulum stress response in immunity and autoimmunity[END_REF]. UPR induces several pathways that lead to a reduced protein load that enters the ER, to increase the ability of the ER to handle unfolded proteins and in some circumstances to trigger cell death [START_REF] Ron | Signal integration in the endoplasmic reticulum unfolded protein response[END_REF]. In the intestine disregulation of normal ER responses are particularly sensitive since dysfunction of UPR leads to spontaneous colitis in rodents lacking the transcription factor XBP1 (X-box-binding protein-1), involved in protein load [START_REF] Kaser | XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease[END_REF][START_REF] Kaser | Endoplasmic reticulum stress in the intestinal epithelium and inflammatory bowel disease[END_REF]. In Winnie mice with increased intestinal ER stress caused by a missense mutation of the Muc2 mucin gene, GCs suppress ER stress and activation of the UPR in a glucocorticoid receptor-dependent manner [START_REF] Das | Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins[END_REF]. GCs promote correct folding of secreted proteins and facilitate the enhanced removal of misfolded proteins from the ER through up-regulation of genes encoding chaperones and elements of ERassociated degradation (ERAD) [START_REF] Das | Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins[END_REF]. These results suggest that epithelial steroidogenesis may also influence gut homeostasis by modulating ER responses in response to exogenous and endogenous stresses.

Metabolism and function of other steroids in the gut

The steroidogenesis involves not only the production of GCs but also other steroids such as mineralocorticoids and sex steroids (androgens and estrogens). In parallel to the description of gut production of GCs, the ability of IECs to metabolize sex steroids was assessed. From a chemical point of view, few enzymatic steps are required to obtain androgens and estrogens from common precursors of GCs involving the 3 enzymes17α-hydroxylase/17,20-lyase (P450c17), and the17 -hydroxysteroid dehydrogenase (17 HSD). For estrogens there is an additional enzyme, the aromatase (P450aro) (Figure 1). Early in vitro and in vivo studies observed the expression of enzymes involved in estrogen metabolism in colorectal cancer cell lines and normal gut mucosa [START_REF] Sano | 17 beta-Hydroxysteroid dehydrogenase type 2 expression and enzyme activity in the human gastrointestinal tract[END_REF][START_REF] Fiorelli | Estrogen synthesis in human colon cancer epithelial cells[END_REF][START_REF] Fiorelli | Estrogen metabolism in human colorectal cancer cells[END_REF][START_REF] English | Estrogen metabolism and malignancy: analysis of the expression and function of 17beta-hydroxysteroid dehydrogenases in colonic cancer[END_REF][START_REF] Sato | Aromatase in colon carcinoma[END_REF]. Evidence of estrogen production by the IECs in healthy humans was then assessed leading to the observation of higher mucosal production of estrone as compared to estradiol [START_REF] Sato | Steroid sulfatase and estrogen sulfotransferase in colon carcinoma: regulators of intratumoral estrogen concentrations and potent prognostic factors[END_REF].

An important aspect of estrogens is their synthesis in diverse tissues. This may lead to variable exposition of IECs by estrogens, depending on sex or diet, in addition to their own production in the intestinal mucosa. The 17 -hydroxysteroid dehydrogenase (17-HSD) family acts in key steps of the synthesis of sex steroid hormones, mainly of active estrogens and androgens [START_REF] Oduwole | 17Beta-hydroxysteroid dehydrogenase type 2: independent prognostic significance and evidence of estrogen protection in female patients with colon cancer[END_REF]. Several isoforms of the 17-HSD enzyme with diverse enzymatic activities have been characterized in humans and were thoroughly investigated in human colonic mucosa, which mainly express isoform 2 and 4 [START_REF] English | Estrogen metabolism and malignancy: analysis of the expression and function of 17beta-hydroxysteroid dehydrogenases in colonic cancer[END_REF]. These isoforms catalyze the oxidative transformation of estradiol and testosterone to estrone and androstenedione, respectively, which is thus considered to play an important role in the peripheral inactivation of estrogen and androgen [START_REF] Sano | 17 beta-Hydroxysteroid dehydrogenase type 2 expression and enzyme activity in the human gastrointestinal tract[END_REF][START_REF] Fiorelli | Estrogen metabolism in human colorectal cancer cells[END_REF][START_REF] Oduwole | 17Beta-hydroxysteroid dehydrogenase type 2: independent prognostic significance and evidence of estrogen protection in female patients with colon cancer[END_REF][START_REF] Oduwole | Downregulation of estrogen-metabolizing 17 beta-hydroxysteroid dehydrogenase type 2 expression correlates inversely with Ki67 proliferation marker in colon-cancer development[END_REF].

Similarly to GCs, estrogens metabolism (mainly estradiol) seems to occur predominantly in the intestinal crypts of the colon and may thereby exert a significant effect on colonic epithelial cell growth [START_REF] Sano | 17 beta-Hydroxysteroid dehydrogenase type 2 expression and enzyme activity in the human gastrointestinal tract[END_REF][START_REF] Narayan | Estradiol is trophic for colon cancer in mice: effect on ornithine decarboxylase and c-myc messenger RNA[END_REF]. Of interest in this regard is the observation that ovariectomy appears to induce colonic crypt atrophy [START_REF] Hoff | Effect of estrogen on cell proliferation in colonic mucosa of the mouse[END_REF]. Estrogen receptor-(ER-) is highly expressed in gut epithelial cells [START_REF] Campbell-Thompson | Expression of estrogen receptor (ER) subtypes and ERbeta isoforms in colon cancer[END_REF][START_REF] Konstantinopoulos | Oestrogen receptor beta (ERbeta) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour's dedifferentiation[END_REF] and ER- signaling is involved in cellular homeostasis and maintenance of the colonic epithelial barrier function [START_REF] Wada-Hiraike | Role of estrogen receptor beta in colonic epithelium[END_REF][START_REF] Looijer-Van Langen | Estrogen receptor-beta signaling modulates epithelial barrier function[END_REF]. Disruption of ER- expression in ER- KO mice was notably associated with an increase in epithelial cell proliferation, and a decrease of apoptosis and the expression of cellular adhesion molecules [START_REF] Wada-Hiraike | Role of estrogen receptor beta in colonic epithelium[END_REF].

Involvement of intestinal steroidogenesis in pathological disorders

Inflammatory Bowel Diseases

Inflammatory Bowel Diseases (IBDs), namely ulcerative colitis (UC) and Crohn's disease (CD) are chronic, complex, relapsing immune-mediated disorders of the gastrointestinal tract of unknown etiology. Emerging evidence suggests that disease development involves a deregulated immune response to the gut microbiome in genetically susceptible individuals, triggered by environmental factors [START_REF] Baumgart | Inflammatory bowel disease: cause and immunobiology[END_REF][START_REF] Baumgart | Inflammatory bowel disease: clinical aspects and established and evolving therapies[END_REF][START_REF] Baumgart | Crohn's disease[END_REF][START_REF] Ordas | Ulcerative colitis[END_REF]. The expression of CYP11A1, CYP11B1 and LRH-1 were first shown to be decreased in inflamed colon biopsies of CD and UC patients compared to non-inflamed biopsies of IBD patients and control subjects [START_REF] Coste | LRH-1mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease[END_REF]. These observations strongly suggest that impaired production of intestinal GCs might participate in the pathogenesis of IBD. This notion was recently newly strengthened by our recent report [START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF]. While UC and CD share some clinical manifestations, they are distinct pathophysiological diseases. For instance, in contrast to the characteristic transmural lesions that can affect all the intestinal layers in CD patients, inflammation during UC is restricted to colonic mucosa and sub-mucosa. Moreover, recent studies suggest that IEC dysfunction may critically contribute to the pathogenesis of UC [START_REF] Danese | Ulcerative colitis[END_REF][START_REF] Anderson | Metaanalysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47[END_REF]. Our recent work demonstrated that colonic epithelial cells (CEC) isolated from surgical specimens of UC patients have a decreased expression of LRH-1 and some steroidogenic enzymes that ultimately result in a cortisol production deficiency. This impairment of epithelial steroidogenesis was correlated with a decrease of the anti-inflammatory factor PPAR [START_REF] Bouguen | Intestinal steroidogenesis controls PPARgamma expression in the colon and is impaired during UC[END_REF]. This study demonstrates complete steroidogenesis and cortisol production in normal human CECs, highlighting a new component of intestinal epithelial barrier regulation. In addition, these results enlarge the GC mechanism of action by suggesting a new anti-inflammatory role of corticosteroids via the induction of PPAR expression in IECs. These findings also support the notion that intestinal production of GCs is a key element in the control of intestinal homeostasis and that the defective synthesis of cortisol by CECs may actively contribute to the pathogenesis of UC.

During IBD two other circumstances may involve intestinal steroidogenesis. We discussed above the possible impact of GCs on ER stress. Induction of ER stress in intestinal epithelium through tissue (and cell type)-specific disruption of XBP1 was shown to result in spontaneous enteritis due to inability of XBP1-deficient IECs to properly generate antimicrobial activity and respond appropriately to inflammatory signals in the local milieu [START_REF] Kaser | XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease[END_REF]. This concept was translated to patients with IBD, who are more likely to develop both CD and UC when bearing single nucleotide polymorphisms within the XBP1 gene locus [START_REF] Kaser | XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease[END_REF]. The steroidogenesis state in patients with CD is not yet known. Since GCs are able to relieve the ER stress [START_REF] Das | Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins[END_REF], UPR are more likely to be impaired in UC patients with defective IEC synthesis of cortisol.

Another side of steroidogenesis implies sex hormones, particularly estrogens. In two large prospective cohorts of women a significant association between oral contraceptive use and risk of CD were also associated with a modest, but non-significant increase in risk of UC [START_REF] Khalili | Oral contraceptives, reproductive factors and risk of inflammatory bowel disease[END_REF].

Conversely postmenopausal hormone use was associated with an increased risk of UC but not CD. As discussed previously, estrogen signaling is involved in the colonic barrier function [START_REF] Wada-Hiraike | Role of estrogen receptor beta in colonic epithelium[END_REF][START_REF] Looijer-Van Langen | Estrogen receptor-beta signaling modulates epithelial barrier function[END_REF]. Decreased estrogen receptor mRNA levels were observed in colonic biopsies from IBD patients as well as in IL-10 deficient mice (a model of IBD), which correlated with an increase in epithelial permeability [START_REF] Looijer-Van Langen | Estrogen receptor-beta signaling modulates epithelial barrier function[END_REF]. These findings indicate a potential impact of sex hormones, in particular estrogens, on IBD development. However, to our knowledge no study has thus far assessed estrogen synthesis in the intestinal mucosa of IBD patients

Colorectal cancer (CRC)

Epidemiological and observational studies pointed out a correlation of exogenous and endogenous estrogens in the development of colorectal cancer (CRC) with a possible increased risk in women with higher circulating endogenous estrogen concentrations [START_REF] Foster | Oestrogen and colorectal cancer: mechanisms and controversies[END_REF].

Estrogens may induce colorectal cancer cell apoptosis by binding to estrogen receptor , [START_REF] Fiorelli | Estrogen synthesis in human colon cancer epithelial cells[END_REF][START_REF] Wilkins | Estrogen prevents sustained COLO-205 human colon cancer cell growth by inducing apoptosis, decreasing c-myb protein, and decreasing transcription of the anti-apoptotic protein bcl-2[END_REF]. A2-2.4-fold increased of estrogen (mainly estrone rather than estradiol) were observed in colon carcinoma tissues [START_REF] Sato | Steroid sulfatase and estrogen sulfotransferase in colon carcinoma: regulators of intratumoral estrogen concentrations and potent prognostic factors[END_REF]. This may be related to overexpression of enzymes involved in steroid synthesis pathway, such as sulfatase and mainly 17HSD isoform 2 [START_REF] Sato | Steroid sulfatase and estrogen sulfotransferase in colon carcinoma: regulators of intratumoral estrogen concentrations and potent prognostic factors[END_REF][START_REF] Oduwole | Downregulation of estrogen-metabolizing 17 beta-hydroxysteroid dehydrogenase type 2 expression correlates inversely with Ki67 proliferation marker in colon-cancer development[END_REF]. The 17HSD2 catalyzes the oxidative transformation of estradiol to estrone [START_REF] Fiorelli | Estrogen metabolism in human colorectal cancer cells[END_REF]. Both the level of estradiol and 17HSD have been associated with CRC and disease progression irrespective of sex. Since IECs are a source of estrogen, the control of epithelial steroidogenesis could be useful to control CRC onset and progression.

Another hypothetical involvement of IEC steroidogenesis may be via PPAR . Beside the antiinflammatory properties of PPAR this nuclear receptor regulates cell proliferation, differentiation and is able to induce apoptosis [START_REF] Han | Peroxisome proliferator-activated receptor gamma: a novel target for cancer therapeutics?[END_REF]. Activation of PPAR by a synthetic ligand (mesalasine) has been shown to inhibit cancer growth by decreasing cell proliferation and increase cell apoptosis [START_REF] Schwab | PPARgamma is involved in mesalazine-mediated induction of apoptosis and inhibition of cell growth in colon cancer cells[END_REF]. Since a decrease of PPAR expression has been observed in some patients bearing CRC [START_REF] Feilchenfeldt | Peroxisome proliferatoractivated receptors (PPARs) and associated transcription factors in colon cancer: reduced expression of PPARgamma-coactivator 1 (PGC-1)[END_REF], a decrease in local GCs production in these patients may be involved, but no data are presently available. On the other hand, it has been demonstrated that CRC cell lines and primary tumor samples produce and release cortisol that can potentially modulate T cell-dependent anti-tumoral immune response, thereby contributing to tumor immune escape [START_REF] Sidler | Colon cancer cells produce immunoregulatory glucocorticoids[END_REF].

Conclusion

Steroid metabolism has been demonstrated and confirmed in the intestine. IECs are able to de novo synthesize and release GCs that regulate the gut immune response. The defective synthesis of cortisol identified in the colonic epithelial cells of UC patients strengthens the idea that intestinal GCs contribute to intestinal homeostasis. In addition, perturbations of estrogen metabolism are also evident in CRC cells, strongly suggesting that steroid metabolism is an important process during IEC homeostasis.

Whereas in classical steroidogenic organs (adrenals and gonads), the regulation of steroid production is well characterized, a lot of work remains to be done in order to identify the mechanisms controlling intestinal steroidogenesis. For instance, the recent identification of microbiota as a potential regulator of corticosterone production by the small intestine in mice [START_REF] Mukherji | Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs[END_REF] may pave the way to design new studies that aim to more thoroughly characterize the production of GCs in the gut. Given the involvement of steroids produced by the gut in pathological conditions such as CRC and IBD, therapeutic strategies targeting intestinal steroidogenesis might represent interesting new approaches for the treatment of these chronic diseases. Glucocorticoids and sex steroids result from the conversion of cholesterol by cytochrome P450 enzymes as well as dehydrogenase enzymes (hydroxysteroid dehydrogenases; HSD).

FIGURE LEGEND

StAR protein is a transporter involved in the cholesterol transport to the mitochondria where the first, rate-limiting step catalyzed by P450scc occurs. In humans, cortisol can also result from the conversion of the metabolically inactive pro-hormone cortisone. In rodents, the first half of the synthesis pathway is conserved, except that rodents do not express the cyp17a1 gene in adrenals. Progesterone is transformed into 11-deoxycorticosterone (instead of 11deoxycortisol) and 11-deoxycorticosterone is transformed into corticosterone (instead of cortisol). Corticosterone can also results from the conversion of 11-dehydrocorticosterone (not represented). For each step, both the name of the protein and the corresponding gene are indicated, with the name of the gene in parenthesis.

  . The development of a human intestinal xenograft model demonstrated the responsiveness of the human small intestine to cortisone during the maturation period [52, 53]. More recently, in vitro studies demonstrated the role of GCs in the maintenance of intestinal epithelial barrier integrity [54]. Boivin et al. used a human intestinal epithelial monolayer model consisting of filter-grown Caco-2 cells to investigate the role of GCs on intestinal barrier function. By measuring transepithelial electric resistance (TEER)

Figure 1 :

 1 Figure 1: A simplified overview of the cortisol and sex steroids synthesis pathway.

Acknowledgement

Research from the Pr. Desreumaux's lab on this subject was supported by research grants from Ferring, the Association François Aupetit and BREMICI (Shering-Plough). Research from the Pr. Brunner's lab on this subject was supported by grants from the German Research Foundation. English proofreading services were provided by Rachel Tipton.

Conflict of interest

GB received lecture fees from Ferring, MSD pharma, Abbvie.

PD has the following conflicts of interest for consulting fees, lecture fees or grant supports in the field of PPAR with Giuliani SpA, Milano, Italy, Procter and Gamble, London, UK, Shire Pharmaceuticals, USA and Lesaffre, Marcq en Baroeul, France. LD, TB and BB have no conflict of interest to disclose.