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Characterisation of different stages of hadronic showers using the CALICE Si-W ECAL physics prototype The CALICE Collaboration

Characterisation of different stages of hadronic showers using the CALICE Si-W ECAL physics prototype

Introduction

The design of particle detectors at future high-energy physics experiments and, in particular, at linear colliders is oriented towards the usage of Particle Flow Algorithms (PFA) for the event reconstruction. These algorithms aim to achieve good jet energy resolution by reconstructing individual particles and hence require high granularity calorimeters [START_REF] Brient | The Calorimetry at the future e+ e-linear collider[END_REF][START_REF] Morgunov | Calorimetry design with energy-flow concept (imaging detector for high-energy physics)[END_REF][START_REF] Thomson | Particle Flow Calorimetry and the PandoraPFA Algorithm[END_REF].

The analysis complements studies presented in [START_REF] Adloff | Track segments in hadronic showers in a highly granular scintillator-steel hadron calorimeter[END_REF] and [START_REF]Tracking within Hadronic Showers in the SDHCAL prototype using Hough Transform Technique[END_REF] for tracking in CALICE prototypes of hadronic calorimeters.

The Si-W ECAL physics prototype

The Si-W ECAL physics prototype has a sandwich-like structure comprising 30 layers of silicon as the active material, alternating with tungsten as the absorber material. The active layers are made of Si wafers segmented in 1 × 1 cm 2 pads. As shown in Fig. 1, each wafer consists of a square of 6 × 6 pads and each layer is a matrix of 3 × 3 of these wafers resulting in an active zone of 18 × 18 cm 2 . The Si-W ECAL is subdivided into three modules of ten layers. The tungsten depth per layer is 1.4 mm (0.4 radiation lengths, X 0 ) in the first module, 2.8 mm in the second and 4.2 mm in the third.

The total thickness corresponds to 24 X 0 and about one nuclear interaction length λ I . Therefore more than half of the hadrons are expected have a primary interaction within the detector volume. A more detailed description of the prototype can be found in Ref. [START_REF] Repond | Design and Electronics Commissioning of the Physics Prototype of a Si-W Electromagnetic Calorimeter for the International Linear Collider[END_REF].

For the analysis presented in this article it is convenient to introduce a unit grid based on the Si-W 3 ECAL pad identifiers according to x = (x, y, z) =    x = 0..17 y = 0..17 z = 0..29, [START_REF] Brient | The Calorimetry at the future e+ e-linear collider[END_REF] where pad counting starts in the bottom right pad, see Fig. 1. Distances in this grid are measured in grid units, g.u.

Data and Monte Carlo samples

Experimental setup at FNAL

The test beam measurements were carried out at the Fermilab Test Beam Facility 7 , FTBF, at FNAL in May and July 2008. A schematic overview of the beam line is given in Fig. 2. The Si-W ECAL was placed in front of two other CALICE physics prototypes, the analogue hadronic calorimeter (HCAL) [START_REF] Adloff | Construction and Commissioning of the CALICE Analog Hadron Calorimeter Prototype[END_REF] and a TailCatcher [START_REF]Construction and performance of a silicon photomultiplier/extruded scintillator tail-catcher and muon-tracker[END_REF]. In both steel is used as absorber. Sensors are scintillator tiles (HCAL) or scintillator strips read out by silicon-photomultipliers. The beam-line also included wire chambers (WC1-3), drift chambers (DC1-4) and scintillator counters of different sizes, named T100(A,B), VETO, T20x20 and T10x10(A,B). The latter two cover an area of 10 × 10 cm 2 each and are used for triggering on beam particles analysed for this article. Finally, two Cherenkov detectors for particle identification are located upstream of the Wire Chamber 1. The chosen coordinate system is right-handed with the z-axis pointing along the beam direction and the y-axis being vertical. The data analysed in this article comprise runs with π --mesons with energies of 2, 4, 6, 8 and 10 GeV.

Monte Carlo simulations

Monte Carlo simulations were carried out within the Mokka framework [START_REF] Mora De Freitas | Detector simulation with MOKKA / GEANT4: Present and future[END_REF], which provides the geometry interface to geant4. There are several models of hadronic interactions available within geant4 that are combined into simulation models. Each hadronic interaction model has its own theoretical basis valid mainly in a specific energy range of hadrons. In this analysis, three simulation models contained in geant4 Version 10.1 are compared with the data:

• ftfp bert uses the Bertini Cascade Model [START_REF] Wright | The Geant4 Bertini Cascade[END_REF] and the Fritiof String Model [START_REF] Andersson | A Model for Low p(t) Hadronic Reactions, with Generalizations to Hadron -Nucleus and Nucleus-Nucleus Collisions[END_REF][START_REF] Nilsson-Almqvist | Interactions Between Hadrons and Nuclei: The Lund Monte Carlo, Fritiof Version 1.6[END_REF] where the first is used for hadron energies below and the second for hadron energies above 4.5 GeV; 7 Fermilab Test Beam Facility web page: http://www-ppd.fnal.gov/MTBF-w

• qgsp bert uses the Bertini Cascade Model at energies below, and the Fritiof String Model for energies above 9.9 GeV. The Fritiof String Model replaces the LHEP parametrisation that was employed until Version 9.6 of geant4;

• qbbc uses also the Bertini Cascade Model for energies below and the Fritiof String Model for energies above 9.9 GeV but interpolates in a larger transition region (for protons and neutrons below 1.5 GeV the Binary Cascade Model [START_REF] Folger | The Binary Cascade[END_REF] is used).

The validity ranges of hadronic interaction models in the three simulation models are illustrated in Fig. 3. More information about these and other simulation models can be found in Ref. [START_REF]Reference Physics Lists[END_REF].

Event selection and preprocessing

The FNAL π -test beam is contaminated with µ -and e -, in particular at lower energies. At 2

GeV the beam contains about 5% π --mesons and 70% electrons. Events are triggered using the signals from the two scintillator counters T10x10A and T10x10B upstream of the Si-W ECAL and π --mesons are identified by using Cherenkov counters. The response of the Si-W ECAL to charged particles was calibrated with a 32 GeV µ -beam [START_REF] Li | Higgs Recoil Mass and Cross-Section Analysis at ILC AND Calibration of the CALICE SiW ECAL Prototype[END_REF] and the deposited energy is converted into units of most probable energy depositions, called MIP hereafter, by particles with behaviour that is approximately minimally ionising. The deposited energy measured in a pad is called hit.

To select π --meson showers, data and simulation samples are required to satisfy similar criteria to those of Refs. [START_REF] Bilki | Testing hadronic interaction models using a highly granular silicon-tungsten calorimeter[END_REF][START_REF] Doublet | Hadrons in a highly granular SiW ECAL -Top quark production at the ILC[END_REF], as below:

• Selection criteria are applied to reject multi-particle events caused by beam impurities or products of decays or upstream interactions of beam particles;

• A lower threshold of 0.6 MIP is chosen to remove noise hits in the Si-W ECAL;

• A hit is classified as being isolated if all the 26 pads in the surrounding cube (in g.u.) have no signal above the noise threshold. The analysis presented in this article uses the non-isolated hits that remain after this removal. The term 'hits' will continue to be used to indicate only non-isolated hits in the following.

• A total of at least 25 hits in the Si-W ECAL is required to remove particles with large incident angle;

• For the event selection the hit coordinates x hit and y hit are defined in the coordinate frame according to Fig. 2. The barycentres of the transverse coordinates xhit and ȳhit of the hits are calculated as: xhit = hits

x hit E hit hits E hit and ȳhit = hits

y hit E hit hits E hit , (2)
where E hit is the energy of a hit in MIP units, and the sums run over all hits in the calorimeter.

The event is accepted if -50 mm < xhit < 50 mm and -50 mm < ȳhit < 50 mm to reduce lateral shower leakage;

• Initially, the interaction layer i is identified as the first of three consecutive layers for which

E i > E cut , E i+1 > E cut and E i+2 > E cut . (3)
with E i being the total energy of layer i;

This simple condition is inefficient at low energies and is extended by the following relative energy increase

E i + E i+1 E i-1 + E i-2 > F cut and E i+1 + E i+2 E i-1 + E i-2 > F cut . (4)
The variables E cut and F cut are free parameters with empirical values of eight MIP and six, respectively. It is argued in [START_REF] Bilki | Testing hadronic interaction models using a highly granular silicon-tungsten calorimeter[END_REF] and references therein that these values optimise the selection efficiency in the energy range relevant for the present study. The event is selected if 5 < i < 15 to suppress electron contamination and to ensure secondaries that extend over several layers after the interaction.

The track-finding algorithm

The track-finding algorithm reconstructs forward-scattered tracks from the interaction between the π --meson and the absorber material in the absence of a magnetic field.

The algorithm consists of three stages:

• identification and removal of interaction region;

• clusterisation of energy deposits;

• formation of track-like clusters;

The entire algorithm is carried out in the grid units introduced in Sec. 2.

Identification and removal of the interaction region

A typical inelastic hadronic interaction in the Si-W ECAL creates a shower with an interaction region and tracks of long-lived particles emerging from it. The interaction region is created by particles such as electrons, photons and low-momentum hadrons that have a short distance of flight in the absorber material of the Si-W ECAL.

In the present analysis the interaction region is defined by all hits that have at least six neighbouring pads with a signal above the noise threshold. For the minimal value of six pads a interaction region is wrongly identified in only 1% of single muon events. Muon events are used to estimate the fraction of events in which this procedure incorrectly identifies an interaction region. For the minimal value of six (five) pads, an interaction region is found in 1% (10%) of muon events. Increasing the minimal value to seven neighbouring pads with hits further reduces the fraction of events with a fake interaction region but does not alter the results presented below in Sec. 5. Figure 4a displays an event after applying noise and isolated hits filters and Fig. 4b is the same event after removal of the interaction region, illustrating that the interaction region is the starting point for secondary tracks.

Clusterisation of energy deposits

During the clusterisation step the energy deposits that are not assigned to the interaction region are grouped into clusters according to topological criteria. The steps of the clusterisation algorithm are described below, with reference to Fig. 5.

1. The separation of tracks improves with increasing distance from the interaction layer. Therefore, the search for hits to seed a cluster begins in the layers that have largest z and continues in the direction of decreasing z. Typically, seeding hits are found in the last layer of the detector; 2. A hit can only be attributed to one cluster. This condition excludes double counting of hits. A random choice of seeding hits shows that effects arising from ambiguities in the assignment of hits to clusters such as the order in which clusters are created, are negligible; 3. For the clusterisation a nearest-neighbour clustering scheme is applied where for each newly associated hit with coordinates (x n , y n , z n ), the algorithm finds nearby hits with the following conditions:

• a neighbour hit should have a z coordinate within [z n -2, z n ] g. u. ;

• the transverse coordinates of neighbouring hits is searched within ranges

[x n -1, x n + 1] and [y n -1, y n + 1].
The search region for nearby hits is visualised in Fig. 5 as a 'red cube' with 3 × 3 × 3 pads; 4. For each newly associated hit the steps 2 and 3 are repeated until the process reaches the first layer of the calorimeter or until no more neighbour hits are found.

The algorithm is motivated by a maximum correspondence between the number of clusters and the number of detected tracks after classification and merging.

Formation of tracks

Secondary long-lived charged particles from hadronic interactions can leave straight, MIP-like tracks in the detector. The goal of the classification of the clusters obtained in the previous step is thus to select track-like clusters.

The classification algorithm executes the following steps:

1. Calculate the number of hits, N hits , in a cluster and reject all clusters with only two hits as residual noise clusters 2. Calculate the length l ∈ R of the considered cluster as the maximal distance between any pair of hits that are in the cluster. For example the lower cluster in Fig. 5 has a length of

∆y 2 n + ∆z 2 n = √ 25 + 1 ≈ 5.1 g.u. ;
3. Reject a cluster with a length of less than l cut = 2 g.u.. This corresponds to the minimal length of a track-like cluster with 3 hits; 4. Compute the following observable

ξ = l N hits -1 + εN hits , (5)
as a measure for the eccentricity of the cluster. The first term of Eq. 5 is motivated by the linear dependence of N hits -1 on the cluster length l, illustrated in Fig. 6. The second term introduces a free parameter ε as an ad hoc correction to increase the efficiency for selecting clusters that do not match the nominal 'pencil-like' topology, as explained below. The value ε = 0.03 was chosen after visual inspection of a few tens of events for pion energies of 10 GeV in an event display.

The chosen value is a compromise between a too small value at which also muon tracks would get assigned more than one track and too large values at which even for electrons the algorithm would result in one single track. The choice made for 10 GeV is also adequate for the other energies relevant for this analysis. For a detailed discussion see Refs. [START_REF]Tracks of hadronic showers in the Si-W ECAL physics prototype[END_REF] and [START_REF] Bilokin | Hadronic showers in a highly granular silicon-tungsten calorimeter and production of bottom and top quarks at the ILC[END_REF] ;

5. If ξ ≥ 1, a cluster is considered as track-like. Otherwise, the cluster is classified as two inseparable tracks.

Due to effects such as multiple scattering, residual detector-noise, δ-rays or the residual arbitrariness in the assignment of hits to clusters, the reconstructed tracks are in general not exactly pencil-like.

The correction term εN hits in the definition of ξ serves to keep a cluster as track-like even if it has large N hits and its form is not strictly pencil-shaped, i.e. l/(N hits -1) < 1. Figure 6: Correlation between N hits -1 and cluster length l in g.u. for a sample of simulated pion interactions with an energy of 10 GeV using the qgsp bert simulation model. Clusters inside the red parabola are rejected by means of Eq. 5. To guide the eye a black line for N hits -1 = l is also included in the figure .   A cluster is classified as being produced by the incoming π --meson if it starts in the first module of the Si-W ECAL and if it has a small polar angle with respect to the z-axis. An example of a cluster produced by an incoming π --meson is visible in Fig. 4b. Clusters assigned to the incoming π --meson are discarded in the following analysis. The remaining track-like clusters are merged into tracks if the relative angle θ c between these clusters fulfils the condition sin θ c < 0.15, optimised using the polar angle distribution of secondary particles in simulated π --meson interactions.

For a sample of single, isolated 6 GeV muons, the track-finding algorithm finds a single track with a 99.7% efficiency.

Results

Observables characterising the interaction region and secondary particles are measured in data and are compared with predictions of the three geant4 simulation models introduced in Sec. 3.2. The average values of observables are also used to make quantitative comparisons. After pre-selection the data have a residual contamination of 8.8% (1.5%) double π --meson events at 2 GeV (10 GeV) beam energy [START_REF] Bilki | Testing hadronic interaction models using a highly granular silicon-tungsten calorimeter[END_REF]. Therefore, for comparison with data all simulation samples were produced with an admixture of double π -events. For average values of observables, correction factors are extracted for each Monte Carlo sample by comparing the results of contaminated samples with those from pure samples. To account for residual contamination, the averages of the data are multiplied by final correction factors. This final correction factors are given by the arithmetic mean of correction factors of the three considered simulation models. The final correction factors are between 0.99 and 1.01 and their uncertainties are much less than one percent. The total systematic error never exceeds two percent and is dominated by two other sources of systematic uncertainties that have been studied.

These are the lowering of the MIP threshold from the nominal 0.6 to 0.4 and the uncertainty on the absolute MIP energy scale [START_REF] Adloff | Track segments in hadronic showers in a highly granular scintillator-steel hadron calorimeter[END_REF] that has been varied by ±2%.

Energy fraction of the interaction region

The first estimator to characterise the interaction of π --mesons with the absorber material is the fraction

f IR = E IR E tot (6)
where E IR is the energy deposited in the interaction region and E tot is the total energy deposited in the Si-W ECAL .

Figure 7 compares the distribution of f IR in data with the three simulation models. The lowest bin of these histograms corresponds to the fraction of events for which no interaction region is found by the algorithm. The rest of the distribution can be approximately described by a skewed normal distribution. The mean value of f IR is shifted towards larger values in data with respect to simulation.

Qualitatively, this observation suggests a different repartition of the deposited energy between the dense interaction region and the sparser parts of the shower in data and the simulation models.

Figure 8 shows the average value of f IR , f IR , as a function of beam energy for beam energies of 2, 4, 6, 8 and 10 GeV. Only events in which an interaction region has been detected are included in f IR . An increase of f IR with increasing beam energy from 43% to around 64% is observed.

Qualitatively, this is expected as number of particles increases with increasing energy but also the electromagnetic component of the hadronic shower becomes increasingly important for higher energies of the interacting π --meson. All three simulation models underestimate the energy fraction by about 10-15% while the slope is reproduced to a much better level. The average fraction f IR as a function of the beam energy for data (black points with grey shaded error band) and the three simulation models. Error bars represent statistical errors. The error band is the sum in quadrature of the systematic error and the statistical error. Only events for which an interaction region has been detected are included in f IR .

Lateral radius of interaction region

The lateral radius r IR of the detected interaction region is a measure of the spatial extension of the interaction region. It is defined as:

r IR = 1 N IR hits hit∈IR (x IR -x hit ) 2 + (ȳ IR -y hit ) 2 , (7)
where the sum runs over the hits in the interaction region, here labelled by IR, and N IR hits is the number of hits in the interaction region. In Eq. 7 xIR and ȳIR are the transverse coordinates of the barycentre of the interaction region, which in analogy with Eq. 2, are defined as:

xIR = hit∈IR x hit E hit hit∈IR E hit and ȳIR = hit∈IR y hit E hit hit∈IR E hit . (8)
Distributions of r IR for data and the predictions of the three simulation models are displayed in Fig. 9 for π --meson energies of 2 and 10 GeV. In both cases, the measured interaction region is wider than the predictions by the simulation models. Figure 10 displays the dependence of the average r IR , r IR , on the beam energy for the data and the three simulation models. Again, only events in which an interaction region has been detected are included in r IR . The lateral size of the interaction region increases with increasing beam energy. This trend is the same for data and for the simulation models. The interaction region measured in data is about 10% wider than those predicted by the three simulation models, all of which yield similar distributions.

[mm]

Beam energy [GeV]

Number of clusters

The final tracks are composed from segments that are given by clusters, as described in Sec. 4.2.

This motivates studying the total number of clusters, N clusters , reconstructed in each event by the track-finding algorithm. This observable is independent of details of the track-finding algorithm since it depends neither on the ε parameter value nor on other free parameters of the classification algorithm.

Note that in all following discussion, only events in which an interaction region has been detected are considered. Figure 11 compares the distribution of N clusters in data with the predictions of the three geant4 simulation models for incoming π --mesons with energies of 2 and 10 GeV. The data are described well by the simulation albeit being slightly shifted towards higher values.

Figure 12 shows the dependence of the average number of clusters, N clusters , on the beam energy for data and the simulation models. The predictions of the models are systematically below data at all energies. The largest deviation is about 7%. The agreement tends to improve with increasing beam energy and is best at 10 GeV.

Beam energy [GeV]

Number of tracks

The N tracks distributions are given in Fig. 13 for data and the three simulation models for energies of 2 and 10 GeV of the incoming π --mesons, respectively. Data and simulation are in good agreement, although at 10 GeV the simulation predicts a narrower spread in N tracks than data.

Figure 14 shows the dependence of N tracks on the beam energy for data and the simulation models.

With increasing beam energy the centre-of-mass energy available for the π-tungsten scattering increases with the square-root of the beam energy according to fixed target kinematics. It is therefore expected that the number of outgoing tracks increases correspondingly. This is indeed observed in data and simulation. The approximately linear increase at smallest energies flattens out towards higher beam energies. The extension of the interaction zone also increases with energy, see for example Sec. 5.2.

This makes it more and more difficult to reconstruct clean tracks in the finite volume of the Si-W ECAL. The simulation models are in agreement with the data at beam energies of 2 GeV and 10 GeV and underestimate the number of secondary tracks by up to 7% at intervening energies.

Beam energy [GeV]

Number of hits per track

The number of hits per track N t hits is an essential characteristic of the reconstructed tracks. The histograms of N t hits for 2 and 10 GeV beam energy are shown in Fig. 15. The distributions obtained for data and Monte Carlo are in good agreement with each other.

Figure 16 shows the dependence of N t hits on the beam energy for data and the simulation models.

Data and simulation agree within 5%. For energies greater than 4 GeV all simulation models are, however, systematically above the data. Note that the average number of hits slightly decreases with increasing energy. The increasing size of the interaction zone limits the space available for track reconstruction. This observation is, therefore, consistent with the flattening of the number of tracks observed in Sec. 5.4.

Angular distributions

Due to the high granularity of the Si-W ECAL further tracking observables such as the polar (θ) and azimuthal (φ) angles of secondary tracks become available. Both angles are measured in the right-handed coordinate frame defined in Sec. 3.1 with θ measured relative to the z-axis. The track direction is calculated from the position of the first and the last hit of the track along the z-axis.

Figures 17 and 18 display histograms of the φ and θ angles, respectively, for 2 and 10 GeV data together with corresponding corresponding results from simulation models. When corrected for the staggering of the detector layers in x [START_REF] Repond | Design and Electronics Commissioning of the Physics Prototype of a Si-W Electromagnetic Calorimeter for the International Linear Collider[END_REF], the pad coordinates of the Si-W ECAL define a grid with a step width of about 1 cm in the lateral direction. This leads to a discretisation of the measured track direction. In particular, in the case of the azimuthal angle φ, values that are a multiple of π/4 are favoured. Beyond that the distribution in φ is isotropic as expected. The bulk of the tracks are scattered in polar angles θ less than π/2 as expected for a fixed target scattering. On the other hand the polar angle spectrum develops a long tail created by backward scattered particles. The simulation models reproduce the data adequately, largely within the experimental uncertainties.

The truncated mean of the θ angle, θ , which can be interpreted as a measure of the collimation of the secondary particles, is shown in Fig. 19 as a function of the beam energy. Here tracks with polar angles smaller than π/2 have been selected. The observable θ has only a weak dependence on the beam energy but shows the tendency to decrease with increasing energy as expected due to the increase of the boost transferred to the secondary particles. The simulation models reproduce the data within a few percent, albeit the boost is less visible.

[rad] φ

Energy deposition by secondary tracks

At energies relevant for this study, the secondaries that create sizeable tracks cross the detector volume behaving in a similar manner to minimally ionising particles. This fact may be exploited as an in situ calibration of the detector, or at least to monitor the response of individual detector regions.

For this specific study the selection criteria of events and tracks are modified as follows:

• events are required to have more than one track and an interaction region to suppress soft inelastic scattering interactions at lower energies;

• reconstructed tracks must have a length l ≥ 8 g. u. and l/N hits > 0.9 g. u. to select long 'pencil-like' tracks;

• Reconstructed tracks must have a polar angle θ < 0.3 rad to reduce the angular dependence of the energy deposits. Figure 21 presents the dependence of the most probable value (MPV) of the energy deposition in secondary tracks on beam energy. The MPV is about 1.05, which is compatible with the fact that the selected tracks cross the detector pads at a small angle. It can be seen that the detector response is both uniform within 1-2% for the analysed energy range in data and reproduced by the simulation models within 1-2%.

[MIP] The algorithm has selected particles with approximately minimally ionising behaviour. The uniform response supports the idea that the secondary tracks can be used for the in situ monitoring of the calibration.

Summary and outlook

This study reveals the large potential of the CALICE Si-W ECAL physics prototype to obtain a detailed picture of the interactions of hadrons with matter. The article describes basic ideas and the application of a new simple track-finding algorithm for the Si-W ECAL. This algorithm allows for the reconstruction of tracks produced by secondary particles created in the interaction of hadrons with the absorber material, and hence to study the interaction region of hadron showers in the Si-W ECAL. The track-finding algorithm produces a new set of observables, based on reconstructed tracks of secondary particles and the interaction region of the hadronic cascades. Data recorded in test beams at FNAL in 2008 using π --mesons with energies between 2 and 10 GeV are compared with predictions from the simulation models qgsp bert, ftfp bert and qbbc as implemented in geant4 Version 10.1. The agreement between data and simulation varies with beam energy and the chosen physics observable. In most cases data and simulation models agree within 10% without revealing a clear preference for one of the chosen simulation models.

The largest discrepancy between data and the simulation models is observed for the deposited energy in and the radius of the interaction region. The measured energy deposition in the interaction region is up to 15% higher than predicted by the Monte Carlo simulation. The distributions of the number of secondary tracks and the number of hits per track for data are described well by the simulation models. The distribution of the polar angle of the reconstructed tracks in the simulation agrees with data within 3-4 % and the distribution of azimuthal angles is reproduced well by the simulation models in spite of the non-trivial detector geometry.

Future work should aim at transferring the insights about the interaction region and the secondaries emerging from it to the optimisation of Particle Flow Algorithms.

A tighter track selection leads to long tracks by particles that show approximately minimally ionising behaviour. The detector response determined using these tracks is stable to about 1-2% over the tested energy range and shows good agreement with simulation. This observation can be exploited as a starting point for a feasibility study of an in situ calibration (or at least a regular monitoring of the detector) by means of the selected tracks.

Figure 1 :

 1 Figure 1: A schematic view of the Si-W ECAL physics prototype.

Figure 2 :

 2 Figure 2: Plan view of the beam line at FNAL. Distances (not to scale) are in mm.

Figure 3 :

 3 Figure 3: Illustration of the validity ranges of the three tested geant4 hadronic interaction models as contained in geant4 Version 10.1.

( a )

 a Before removing the interaction region. (b) After removing the interaction region.

Figure 4 :

 4 Figure 4: Event display of a π --meson interaction with an energy of 10 GeV (a) before and (b) after removal of the interaction region. Smaller cubes are pads that are part of the interaction region and are not processed by the trackfinding algorithm. In this event the hits in the first ten layers are classified as hits left by the incoming π --meson.

Figure 5 :

 5 Figure 5: Illustration of the clusterisation step. The Si-W ECAL hits are represented by blue cubes, and the search region for adjacent hits is indicated by red cubes. The blue arrows point in the direction of the clusterisation flow.

Figure 7 :

 7 Figure7: The f IR distribution for energies of 2 GeV (a) and 10 GeV (b) of the beam energy as observed in data (points with error bars) and for the three simulation models, qgsp bert, ftfp bert and qbbc. The double π --meson background for each of the three models is also shown. The first bin contains events without a detected interaction region. All histograms are normalised to unit area. Error bars represent statistical uncertainties only.

Figure 8 :

 8 Figure 8:The average fraction f IR as a function of the beam energy for data (black points with grey shaded error band) and the three simulation models. Error bars represent statistical errors. The error band is the sum in quadrature of the systematic error and the statistical error. Only events for which an interaction region has been detected are included in f IR .

Figure 9 :

 9 Figure 9: The r IR distribution for energies of 2 GeV (a) and 10 GeV (b) of the beam energy. Other details follow those of Fig. 7.

Figure 10 :

 10 Figure 10: The average radius r IR as a function of the beam energy. Other details follow those of Fig. 8.

Figure 11 :

 11 Figure 11: Number of clusters for energies of 2 GeV (a) and 10 GeV (b) of the beam energy. Only events for which an interaction region has been detected have been included. Other details follow those of Fig. 7.

Figure 12 :

 12 Figure 12: The average number of clusters N clusters as a function of the beam energy. Other details follow those of Fig. 8.

Figure 13 :

 13 Figure 13: Number of secondary tracks for energies of 2 GeV (a) and 10 GeV (b) of the beam energy. Other details follow those of Fig. 11.

Figure 14 :

 14 Figure 14: The average number of secondary tracks N tracks as a function of the beam energy. Other details follow those of Fig. 8.

Figure 15 :

 15 Figure 15: Number of hits per reconstructed track for energies of 2 GeV (a) and 10 GeV (b) of the beam energy. Other details follow those of Fig. 11.

Figure 16 :

 16 Figure 16: The average number of hits per reconstructed track N t hits as a function of the beam energy. Other details follow those of Fig. 8.

Figure 17 :

 17 Figure 17: Comparison of the azimuthal angle φ of secondary tracks for energies of 2 GeV (a) and 10 GeV (b) of the incoming π --mesons. Other details follow those of Fig. 11.

Figure 18 :

 18 Figure 18: The polar angle θ of secondary tracks for energies of 2 GeV (a) and 10 GeV (b) of the incoming π --mesons. Other details follow those of Fig. 11.

Figure 19 :

 19 Figure 19: The truncated mean polar angle θ of secondary tracks as a function of the beam energy. Only tracks with polar angles less than π/2 have been selected. Other details follow those of Fig. 8.

Figure 20

 20 Figure 20 displays the energy deposition per hit by secondary tracks E t dep for beam energies of 2 and 10 GeV. Both distributions peak at around 1 MIP as expected for straight MIP-like tracks. The overlaid fit is a Landau distribution convolved with a Gaussian resolution function, describing the data well. The tighter selection criteria reduce considerably the event sample at 2 GeV. As a consequence, the statistical uncertainty of the fit is large for the 2 GeV sample.

Figure 20 :Figure 21 :

 2021 Figure 20: Energy deposition by secondary tracks observed in data (points with error bars) and for the three simulation models for beam energies of 2 GeV (a) and 10 GeV (b). The spectra are fit by the convolution of a Landau distribution with a Gaussian resolution function. The double π --meson background for the three models is shown by the grey dashed, dotted and dash-dotted histograms, respectively. All histograms are normalised to unity. Error bars represent statistical uncertainties only.

Acknowledgements

We gratefully acknowledge the DESY, CERN and FNAL managements for their support and hospitality, and their accelerator staff for the reliable and efficient beam operation. This work was supported by the FWO, Belgium; by the Natural Sciences and Engineering Research Council of Canada; by the Ministry of Education, Youth and Sports of the Czech Republic; by the P2IO LabEx in the framework 'Investissements d'Avenir' managed by the French National Research Agency (ANR) under Grant Agreements ANR-10-LABX-0038 and ANR-11-IDEX-0003-01; by the 'Quarks and Leptons' Programme of CNRS/IN2P3 France; by the Alexander von Humboldt Stiftung (AvH), Germany; by the Bundesministerium für Bildung und Forschung (BMBF), Germany; by the Deutsche Forschungsgemeinschaft (DFG), Germany; by the Helmholtz-Gemeinschaft (HGF), Germany; by the I-CORE Program of the Planning and Budgeting Committee, Israel; by the Nella and Leon Benoziyo Center for High Energy Physics, Israel; by the Israeli Science Foundation, Israel; by the National Research Foundation of Korea; by the Korea-EU cooperation programme of National Research Foundation of Korea, Grant Agreement 2014K1A3A7A03075053; by the Netherlands Organisation for Scientific Research (NWO); by the Science and Technology Facilities Council, UK; by the Nuclear Physics, Particle Physics, Astrophysics and Cosmology Initiative, a Laboratory Directed Research and Development program at the Pacific Northwest National Laboratory, USA.


Lazaro Emilio Aira

D Goncalves

Jozef P Bossowski

C Rubio-Patino

Johanna Chiche

Rachel Paul-Bellon

L Mondragon

Maéva Gesson

Priscillia Lecucq-Ottavi

Sandrine Obba

Diogo Gonçalves

Camila Rubio-Patiño

Laura Mondragón

Pascal Colosetti

Frédéric Luciano

Béatrice Bailly-Maitre

Laurent Boyer

Arnaud Jacquel

Guillaume Robert

Jean-Ehrland Ricci

Jean-Paul Ortonne

Thierry Passeron

Jean-Philippe Lacour

Patrick Auberger

Sandrine Marchetti
email: marchett@unice.fr

or Pharmacological Inhibition Mitigates Psoriasis-Like Phenotype in Mice

Keywords: Inflammatory response, pro-inflammatory caspases, pro-inflammatory cytokines, keratinocytes, human skin biopsies, psoriasis

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Caspases are a family of proteases (Cysteine-ASPartic proteASES), highly conserved evolutionarily, with a key role in apoptosis and inflammatory signaling pathways [START_REF] Lamkanfi | Caspases in cell survival, proliferation and differentiation[END_REF]. In relation to their biological function, caspases have been classified in apoptotic (2, 3, 6, 7, 8, 9 and 10) and pro-inflammatory (1, 4, 5, 11 and 12) [START_REF] Li | Caspases in apoptosis and beyond[END_REF], mainly involved in cell death signaling pathways and regulation of cytokine maturation during inflammation, respectively. Nevertheless, pro-inflammatory caspases have been also implicated in pyroptosis, another programmed cell death (Lamkanfi andDixit, 2014, Man and[START_REF] Man | Regulation of inflammasome activation[END_REF]. Although proinflammatory caspases 1 and 12 are functional orthologous between human and mice; caspases 4 and 5, only present in human, are the homologous of mouse caspase 11 [START_REF] Liu | A Mechanistic Understanding of Pyroptosis: The Fiery Death Triggered by Invasive Infection[END_REF].

Pro-inflammatory caspases, firstly produced as inactive zymogens, are activated through multiprotein complexes called inflammasomes after cellular stimulation via engagement of pattern recognition receptors [START_REF] Martinon | The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta[END_REF]. Once activated, inflammatory caspases mediated immune response against infectious stress through the maturation and secretion of pro-inflammatory cytokines such as interleukin (IL)-1β and IL-18. Caspase 1 activation and subsequent IL-1β production have been associated with a large variety of inflammatory and autoimmune diseases (reviewed in [START_REF] Gabay | IL-1 pathways in inflammation and human diseases[END_REF][START_REF] Mcilwain | Caspase functions in cell death and disease[END_REF][START_REF] Patel | Inflammasome Priming in Sterile Inflammatory Disease[END_REF]) such as rheumatoid arthritis, type 2 diabetes [START_REF] Ruscitti | Monocytes from patients with rheumatoid arthritis and type 2 diabetes mellitus display an increased production of interleukin (IL)-1beta via the nucleotidebinding domain and leucine-rich repeat containing family pyrin 3(NLRP3)-inflammasome activation: a possible implication for therapeutic decision in these patients[END_REF] and inflammatory bowel diseases (IBD) [START_REF] Perera | NLRP3 Inhibitors as Potential Therapeutic Agents for Treatment of Inflammatory Bowel Disease[END_REF].

Psoriasis is a chronic auto-inflammatory/autoimmune skin disease [START_REF] Lowes | Immunology of psoriasis[END_REF] characterized by an intense dialogue between keratinocytes and immune cells, affecting 2% of the worldwide population [START_REF] Parisi | Global epidemiology of psoriasis: a systematic review of incidence and prevalence[END_REF]. Psoriasis has been classified according to several clinical manifestations, psoriasis vulgaris being the most common type, whose principal feature is the development of red patches with silver scales called psoriasis plaques [START_REF] Deng | The Inflammatory Response in Psoriasis: a Comprehensive Review[END_REF]. A large and complex network of several proinflammatory cytokines such as IL-1-α, IL-1β, IL-17, IL-18, IL-22, IL-23, interferon (IFN)-γ and tumor necrosis factor (TNF)-α have been associated with the development and establishment of psoriasis plaques [START_REF] Nickoloff | The cytokine and chemokine network in psoriasis[END_REF], highlighting a key role of these cytokines in the pathogenesis of psoriasis.

Albeit the role of IL-1 and IL-18 cytokines has been well described in psoriasis pathogenesis [START_REF] Companjen | Elevated interleukin-18 protein expression in early active and progressive plaque-type psoriatic lesions[END_REF][START_REF] Rabeony | IMQ-induced skin inflammation in mice is dependent on IL-1R1 and MyD88 signaling but independent of the NLRP3 inflammasome[END_REF][START_REF] Shimoura | Interleukin (IL)-18, cooperatively with IL-23, induces prominent inflammation and enhances psoriasis-like epidermal hyperplasia[END_REF][START_REF] Singh | Monocyte-derived inflammatory Langerhans cells and dermal dendritic cells mediate psoriasis-like inflammation[END_REF][START_REF] Uribe-Herranz | IL-1R1 signaling facilitates Munro's microabscess formation in psoriasiform imiquimod-induced skin inflammation[END_REF], little is known about the contribution of pro-inflammatory caspases in this context. It has been showed that activated caspase 1 is increased in lesional skin samples from psoriasis patients compared to non-lesional samples [START_REF] Johansen | The activity of caspase-1 is increased in lesional psoriatic epidermis[END_REF][START_REF] Marchetti | The caspasecleaved form of LYN mediates a psoriasis-like inflammatory syndrome in mice[END_REF]. Moreover, an increase in the mRNA expression of caspase 5 was found in lesional biopsies [START_REF] Salskov-Iversen | Caspase-5 expression is upregulated in lesional psoriatic skin[END_REF] and a constitutive expression of this caspase was observed in the epidermis of normal skin [START_REF] Zwicker | Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation[END_REF]. However, the contribution of inflammatory caspases in psoriasis pathogenesis is controversial as Cho et al reported a decrease in the epidermal thickness associated with a significant reduction in IL-1β levels in caspase 1 -/-mice treated with IMQ [START_REF] Cho | IL-17 and IL-22 enhance skin inflammation by stimulating the secretion of IL-1beta by keratinocytes via the ROS-NLRP3caspase-1 pathway[END_REF] whereas another recent study concluded that the invalidation of caspase 1 had no impact in reducing the psoriasis-like phenotype after IMQ treatment [START_REF] Rabeony | IMQ-induced skin inflammation in mice is dependent on IL-1R1 and MyD88 signaling but independent of the NLRP3 inflammasome[END_REF].

Therefore, we decided to address the implication of pro-inflammatory caspases in psoriasis pathogenesis. We first find in a large cohort of psoriasis patient biopsies that caspase 1 and 5 are activated in all lesional samples and that caspase 4 is activated in most lesional biopsies.

Further, caspase 1/11 deficiency and pharmacological inhibition of pro-inflammatory caspases significantly reduce skin inflammatory disease in three mouse models of psoriasis like-phenotype. Moreover, stimulation of normal human keratinocytes (NHK) with TNF-α and IL-17A demonstrates that keratinocytes are primed to activate caspase 5 and secrete IL-1β. Finally, by adoptive transfer experiments we show that pro-inflammatory caspases activation in immune cells is sufficient to induce a complete inflammatory response in mice.

Results

Pro-inflammatory caspases are activated in lesional skin biopsies from psoriasis patients

Although increased mRNA expression of pro-inflammatory caspases 1, 4 and 5 in lesional biopsies from psoriasis patients has been already observed [START_REF] Salskov-Iversen | Caspase-5 expression is upregulated in lesional psoriatic skin[END_REF][START_REF] Zwicker | Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation[END_REF] and expression of cleaved caspase 5 could be detected in one psoriasis patient [START_REF] Zwicker | Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation[END_REF], we decided to assess on a large cohort of psoriasis patients, the expression of caspases to confirm their activation status. To do that, western blot analysis of skin extracts was performed from non lesional and lesional biopsies. Pro-caspase 1, 4 and 5 were all expressed in non-lesional biopsies; however only a strong increase of pro-caspase 5 was observed in lesional skin as compared to non-lesional paired and healthy donor skin (Figure 1a,b,d;Figure S1a). Interestingly, high levels of cleaved caspase 1 and 5 were observed in lesional skin from all analyzed patients (Figure 1a-d; Figure S1a) and cleaved caspase 4 was seen in most analyzed lesional biopsies (Figure 1a,b,d; Figure S1a). Moreover, an increase in inflammasome component expression along with the mature forms of IL-1β and IL-18 was found (Figure S1b), as previously reported [START_REF] Dombrowski | Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions[END_REF][START_REF] Johansen | The activity of caspase-1 is increased in lesional psoriatic epidermis[END_REF]. Altogether, these data suggest that pro-inflammatory caspase activity is regulated in injured skin from psoriasis patients.

Deficiency in pro-inflammatory caspases 1/11 impaired the psoriasis-like disease development in different mouse models

While the role of inflammasome-processed cytokines IL-1β [START_REF] Feldmeyer | Interleukin-1, inflammasomes and the skin[END_REF] and IL-18 [START_REF] Companjen | Elevated interleukin-18 protein expression in early active and progressive plaque-type psoriatic lesions[END_REF]) is well-known in psoriasis, the role of proinflammatory caspases in this scenario is controversial. Therefore, to clearly define the role of pro-inflammatory caspases in psoriasis-like disease, we decided to use three different mouse models: i) the Lyn∆N transgenic mice [START_REF] Marchetti | The caspasecleaved form of LYN mediates a psoriasis-like inflammatory syndrome in mice[END_REF], which develop spontaneously psoriasis after birth; ii) the IMQ-induced skin dermatitis [START_REF] Van Der Fits | Imiquimodinduced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis[END_REF], and iii) the IL-23 intradermal ear injections [START_REF] Hedrick | CCR6 is required for IL-23-induced psoriasis-like inflammation in mice[END_REF].

First, we used the Lyn∆N mice [START_REF] Marchetti | The caspasecleaved form of LYN mediates a psoriasis-like inflammatory syndrome in mice[END_REF] which recapitulate the main features of human psoriasis, as illustrated by the increased skin expression of the principal cytokines implicated in psoriasis pathogenesis (Figure S2a). Interestingly, an increased mRNA expression of some inflammasome components, such as caspase-11, Absent In Melanoma (AIM)-2 and NLRP-3 (Figure S2b), associated to an increase in IL-1β expression both at the mRNA and protein level was observed (Figure S2b,c). Next, we crossed Lyn∆N mice with C1/C11 deficient mice [START_REF] Kuida | Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme[END_REF] to obtain Lyn∆N C1/C11 -/-mice, named hereafter Lyn∆N dKO mice. Strikingly, the absence of pro-inflammatory caspases hampered the skin inflammatory phenotype developed by Lyn∆N mice over time, based on the adapted PASI score that we established (Figure 2a; Figure S3a), producing a significant delay in the initiation of the psoriasis-like phenotype in Lyn∆N dKO mice. Moreover, both the no weight gain linked to the onset of the disease and the reported death of Lyn∆N mice [START_REF] Marchetti | The caspasecleaved form of LYN mediates a psoriasis-like inflammatory syndrome in mice[END_REF] were significantly reduced in Lyn∆N dKO mice (Figure S3b; Figure 2b). The improved phenotype observed in Lyn∆N dKO mice was associated to a decrease in epidermal hyperplasia (Figure 2c,d), along with a reduction in the mRNA expression of inflammasome components such as AIM-2 and NLRP-3 and several pro-inflammatory cytokines (Figure 2e; Figure S3c), with an exception for IL-23 and TNF-α, for which similar levels were obtained.

Concomitantly, the absence of pro-inflammatory caspases significantly reduced the levels of both IL-1β and IL-18 protein expression observed in Lyn∆N mice skin (Figure 2f). Therefore, activation of caspase 1/11 is necessary for the development of a complete inflammatory skin disease in the Lyn∆N mice model.

To confirm the contribution of pro-inflammatory caspases in psoriasis pathogenesis, we induced a psoriasis-like phenotype either by topical IMQ cream application onto the back skin (Figure 3) or by IL-23 intradermal injections into ears (Figure S4) of WT and C1/C11 deficient mice, hereafter named dKO mice. In both models (IMQ and IL-23), the absence of pro-inflammatory caspases delayed the onset of the clinical score measured. Indeed, erythema, scaling and cumulative scores quantified in the IMQ model (Figure 3a) and ear thickness measured in the IL-23 model (Figure S4a,b) were significantly reduced in dKO mice compared to WT mice. Further, H&E staining of skin sections revealed a decrease in epidermal hyperplasia (Figure 3b,c;Figure S4c,d), along with a reduction in the inflammatory cell infiltration (Figure 3d; Figure S4e), with a reduced number of CD45.2 + immune cells into the dKO mice skin, evidenced more precisely by a significant decrease number of inflammatory monocytes and neutrophils. Additionally, inflammasome components, proinflammatory cytokine mRNA expression (Figure 3e; Figure S4f; Figure S5) and IL1β protein level were significantly reduced in dKO mice in both models (Figure 3f; Figure S4g). Surprisingly, IL-18 protein level was only differentially modulated in the IMQ model (Figure 3f). Altogether, these data indicate that regulation of pro-inflammatory caspase activity is necessary to produce an inflammatory response in psoriasis.

Pharmacological inhibition of caspases 1/11 by AC-YVAD-CMK compound reduces IMQ-induced psoriasis like phenotype in mice

It has been previously demonstrated in mice suffering from acute gastric injury that treatment with the selective caspase 1 inhibitor AC-YVAD-CMK led to a protection through the attenuation of NLRP-3 inflammasome activity [START_REF] Zhang | The caspase-1 inhibitor AC-YVAD-CMK attenuates acute gastric injury in mice: involvement of silencing NLRP3 inflammasome activities[END_REF]. Therefore, we next decided to analyze the effect of AC-YVAD-CMK treatment in the IMQ model. To do that, WT mice were treated with IMQ cream together with either DMSO or AC-YVAD-CMK compound injected intra-peritoneally.

While the co-treatment had no additive effect on both weight loss (Figure S6a) and splenomegaly induced by IMQ treatment (Figure S6b) as reported [START_REF] Van Der Fits | Imiquimodinduced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis[END_REF], a significant reduction in erythema, scaling and cumulative score was achieved in mice treated with the selective caspase 1 inhibitor (Figure 4a). It is worth noting that the delay observed upon pharmacological inhibition of caspase 1 started later compared to deficient mice for C1/11 (dKO mice) (Figure 3a). However, the skin phenotype attenuation was associated with a significant decrease in epidermal hyperplasia (Figure 4b,c) and mRNA expression of different inflammasome components and pro-inflammatory cytokines (Figure 4d; Figure S6c).

Additionally, the pharmacological inhibition of caspase 1 was linked to a significant decrease in IL-1β and IL-18 protein levels (Figure 4e,f).

Next, we assessed the AC-YVAD-CMK as a potential therapeutic treatment. To do that, WT mice were first treated with IMQ and then daily treated with four intra-peritoneal injections of AC-YVAD-CMK after two IMQ applications (Figure S6d) and a significant reduction in the induced psoriasis-like disease was observed. Moreover, a trend toward to a significant decrease was found in the pro-inflammatory cytokines and NLRP-3 inflammasome mRNA expression (Figure S6e), which could corroborate the attenuation obtained in the observed phenotype, although no differences in IL-17 mRNA expression were seen.

Altogether these data confirm that pro-inflammatory caspase activation is required for the complete achievement of the inflammatory process implicated in psoriasis pathogenesis.

Primary human keratinocytes are primed to secrete IL-1β in mimicking psoriasis condition in an inflammasome dependent manner

Psoriasis is characterized by a dialogue between keratinocytes in the epidermis and the immune cell infiltration, mainly in the dermis, through the release of cytokines. Thus, to decipher in which skin compartments inflammatory caspases and IL-1β were induced upon IMQ treatment in mice, epidermis/dermis dissociation experiment was performed (Figure S7). A significant increase in IL-1β mRNA expression was found both in epidermis and dermis compartment, while caspase 1 and 11 expression were only increased in the dermis. We next wanted to know whether pro-inflammatory caspases could be activated in normal human keratinocytes (NHK) in vitro upon stimulation with two cytokines implicated in psoriasis pathogenesis: IL-17A and TNF-α [START_REF] Johansen | Characterization of TNF-alphaand IL-17A-Mediated Synergistic Induction of DEFB4 Gene Expression in Human Keratinocytes through IkappaBzeta[END_REF]. As expected, a significant increase in the mRNA expression of IL-6 and IL-8 [START_REF] Bertelsen | IL-17F regulates psoriasis-associated genes through IkappaBzeta[END_REF][START_REF] Fujishima | Involvement of IL-17F via the induction of IL-6 in psoriasis[END_REF] was observed, validating our in vitro model (Figure S8a,b). While no difference in caspase 1 and 4 (Figure 5a,e) expression levels was observed, a significant increase of caspase 5, IL-1β, NLRP-1 and NLRP-3 expression (Figure 5a-d,f), both at the mRNA and protein level, was observed. Moreover, it is interesting to note that there was not only an increase of pro-caspase 5 but also of its activated form at long-term stimulation time points (Figure 5d). Moreover, to know if those primed keratinocytes could be responsible for IL-1β release upon inflammasome activation [START_REF] Stout-Delgado | Impaired NLRP3 inflammasome function in elderly mice during influenza infection is rescued by treatment with nigericin[END_REF], nigericin was added to NHK pretreated for 24h with TNF-α + IL-17A. Interestingly, a significant increase in the release of mature IL-1β and activated caspase 1 was observed (Figure 5g,h), confirming that keratinocytes in a psoriasis environment can be implicated in IL-1β secretion in an inflammatory caspase dependent manner.

Caspase 1/11 activation in immune cells is sufficient for the development of psoriasislike disease in mice

As we showed that 1) the activation of inflammatory caspases is necessary to obtain a complete psoriasis-like disease, and 2) inflammatory caspase activation can take place both in immune cells and keratinocytes, we decided to perform bone marrow transplantation experiments to elucidate in which cell type their activation is responsible for the psoriasis pathogenesis. As expected, dKO mice receiving dKO bone marrow showed a significant decrease in the adapted PASI score (Figure 6a), epidermal hyperplasia (Figure 6b,c), mRNA expression of inflammasome components and pro-inflammatory cytokines (Figure 6d) and IL-1β protein levels (Figure 6e) compared to WT mice transplanted with WT bone marrow. After IMQ treatment, WT mice transplanted with dKO bone marrow showed a significant reduction in erythema and scaling scores (Figure 6a), epidermal hyperplasia (Figure 6b,c), mRNA expression of different pro-inflammatory cytokines (Figure 6d) and IL-1β protein levels (Figure 6e) compared to WT mice, which received WT bone marrow. The reduction in the phenotype observed in this chimeric mouse is comparable to what was observed in dKO mice reconstituted with dKO bone marrow. On the opposite, dKO mice transplanted with WT bone marrow developed a phenotype comparable to WT mice reconstituted with WT bone marrow.

However, although an identical increase in epidermal hyperplasia was obtained (Figure 6b,c), the mRNA expression levels of cytokine and inflammasome components (Figures 6d andS9a) were less pronounced. This discrepancy could be explained by either the absence of radio-sensitive γδ T cells, that require thymocyte transfer to be repopulated (in our study only bone marrow cells were transferred) or the fact that mice lethal irradiation could modify their response to pro-inflammatory challenge.

Nevertheless, altogether these results provided evidence of a major role of inflammatory caspase activation into immune cells rather than keratinocytes to trigger a complete psoriasislike disease. Furthermore, similar results were obtained with the IL-23 mice model (figure S9 b,c), reinforcing the notion that activation of pro-inflammatory caspases in immune cells is sufficient to induce a complete pro-inflammatory response leading to a psoriasis-like phenotype in treated mice. Interestingly, FACs-sorting of immune cells showed that caspase-1 and caspase-11 expression was highly increased in macrophages upon IMQ treatment as compared to granulocytes and CD11c+/CD11b-cells (figure S10), suggesting that it is their expression/activation within macrophages that should be implicated in psoriasis pathogenesis.

However, further experiments are needed to clearly identify in which immune cells inflammatory caspases are activated and required for psoriasis development.

Discussion

Psoriasis is a chronic inflammatory skin disease with an unpredictable course [START_REF] Sabat | Three decades of psoriasis research: where has it led us?[END_REF]. While many efforts have been done to identify molecular factors implicated in the initiation and maintenance of the disease, which allowed the identification of new biological drugs targeting TNF-α, IL17 and IL-23 [START_REF] Lowes | Immunology of psoriasis[END_REF], the etiology of psoriasis is still a matter of concern. In this context, controversial results are found in the literature regarding the impact of inflammasome pathway leading to inflammatory caspase activation and mature IL-1β/IL-18 secretion in psoriasis. Indeed, besides strong demonstration of the implication of IL-1 family members in psoriasis [START_REF] Rabeony | IMQ-induced skin inflammation in mice is dependent on IL-1R1 and MyD88 signaling but independent of the NLRP3 inflammasome[END_REF], the role of inflammatory caspases in this respect is still a matter of debate as Cho et al [START_REF] Cho | IL-17 and IL-22 enhance skin inflammation by stimulating the secretion of IL-1beta by keratinocytes via the ROS-NLRP3caspase-1 pathway[END_REF] and Rabeony et al [START_REF] Rabeony | IMQ-induced skin inflammation in mice is dependent on IL-1R1 and MyD88 signaling but independent of the NLRP3 inflammasome[END_REF] reported opposite results using IMQ psoriasis-like model. However, our results argue in favor of a major role of inflammatory caspases in cytokine maturation in psoriasis as the invalidation or the pharmacological inhibition of pro-inflammatory caspases leads to a significant reduction in the induced inflammatory phenotype in several psoriasis mouse models. Moreover, even if other proteases such as caspase-8 and neutrophil derived proteases have been reported to participate in the maturation of IL-1β and IL-18 [START_REF] Clancy | Neutrophil extracellular traps can serve as platforms for processing and activation of IL-1 family cytokines[END_REF][START_REF] Pierini | AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages[END_REF], our study pointed out inflammatory caspases as major contributors of IL-1β/IL-18 maturation in skin disease. In fact, it has been demonstrated that IL-1β can potentiate the production of IL-17A by γδ-T cells, reinforcing the establishment of the disease [START_REF] Cai | Pivotal role of dermal IL-17-producing γδ T cells in skin inflammation[END_REF][START_REF] Kessel | Proinflammatory Cytokine Environments Can Drive Interleukin-17 Overexpression by γ/δ T Cells in Systemic Juvenile Idiopathic Arthritis[END_REF]. Thus, we can speculate that the significant decrease in IL-1β expression obtained in our study, through the invalidation or inhibition of inflammatory caspases, contributes to attenuate the induced psoriasis-like phenotype by decreasing the secretion of IL-17A by γδ-T cells. This finding is supported by the study of Douglas et al, showing that atopic dermatitis/psoriasis-like disease developed by Sharpin (cpdm) mice, is significantly reduced when caspase 1/11 are deficient [START_REF] Douglas | The Inflammatory Caspases-1 and -11 Mediate the Pathogenesis of Dermatitis in Sharpin-Deficient Mice[END_REF]. Indeed, neither lesions nor epidermal hyperplasia were observed, demonstrating the main role of pro-inflammatory caspases in the induction of dermatitis and the function of inflammasome activation as an initiating signal in Sharpin mice. Interestingly, the delay in the dermatitis onset in Sharpin-C1/C11 -/-mice is consistent with the delay observed in Lyn∆N dKO mice used in the present study, showing the improvement in psoriasis-like phenotype through the invalidation of pro-inflammatory caspases. It is worth noting that while genetic ablation of TNFR1 in Sharpin [START_REF] Rickard | Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation[END_REF] or Lyn∆N [START_REF] Marchetti | The caspasecleaved form of LYN mediates a psoriasis-like inflammatory syndrome in mice[END_REF] mice produced a complete rescue of the phenotype, the invalidation of inflammatory caspases was associated with a delay in the inflammation symptoms, demonstrating that TNF signaling is one of the principal pathway to induce skin inflammation, while inflammatory caspases pathway is more implicated in the maintenance and exacerbation of the disease. Additionally, the level of psoriasis-like phenotype inhibition in the absence of pro-inflammatory caspases is consistent with other studies where other important components for psoriasis disease development, like T cells, are deleted [START_REF] Van Der Fits | Imiquimodinduced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis[END_REF].

Although it was previously demonstrated that cleaved-form of caspase-5 is present in lesional skin biopsies from psoriasis patients [START_REF] Zwicker | Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation[END_REF], we confirmed in a large cohort of lesional and non-lesional skin biopsies that cleaved forms of caspase-1, 4 and 5 are found in all lesional samples of tested psoriasis patients whereas no cleaved forms are found in healthy donor samples. Moreover, cleaved forms of proinflammatory caspases were associated with an increase in the NLRP-3 and AIM-2 inflammasomes. Caspase inhibitors have been evaluated in clinical trials, specifically the small molecule inhibitors VX-740 and VX-765, also known as Pralnacasan and Belnacasan, respectively; which are potent and selective inhibitors of caspase 1 and caspase 4 (Vertex Pharmaceuticals). In our study, we co-treated mice with IMQ and the selective caspase-1 inhibitor AC-YVAD-CMK and a significant decrease in the erythema, scaling and pro-inflammatory cytokine production was found, reinforcing the notion that targeting pro-inflammatory caspases could be of interest to treat psoriasis disease. Indeed, in a model of acute gastric injury, mice were pre-treated with AC-YVAD-CMK and an inhibition in the production of IL-1β was observed leading to the protection of these mice, an effect correlated with the impairment of NLRP-3 inflammasome activity [START_REF] Zhang | The caspase-1 inhibitor AC-YVAD-CMK attenuates acute gastric injury in mice: involvement of silencing NLRP3 inflammasome activities[END_REF]. Moreover, like in AC-YVAD-CMK treated mice, Zhang et al found a strong diminution in other pro-inflammatory cytokines including IL-6, IL-8 and TNFα. Actually, not only caspase inhibitors are evaluated as potential drugs to treat psoriasis disease, but IL-1, IL-18 and inflammasome inhibition could be possible targets to treat inflammatory skin diseases [START_REF] Fenini | Potential of IL-1, IL-18 and Inflammasome Inhibition for the Treatment of Inflammatory Skin Diseases[END_REF]. In this line, psoriasis patients were treated in an open-label clinical trial I/II with the human monoclonal antibody against IL-1α, MABp1, and an encouraging clinical response was achieved that must be improved with an increase in dose/frequency [START_REF] Coleman | Open-Label Trial of MABp1, a True Human Monoclonal Antibody Targeting Interleukin 1alpha, for the Treatment of Psoriasis[END_REF].

In our study, we showed that keratinocytes cultured in conditions mimicking psoriasis (TNFα + IL-17A) are primed to secrete IL-1β in an inflammasome-dependent manner. Recently, it has been demonstrated that the stimulation of keratinocytes with TNF-α or with IL-17A only did not trigger caspase activation [START_REF] Zwicker | Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation[END_REF], but that the combination of at least two pro-inflammatory cytokines is necessary [START_REF] Cho | IL-17 and IL-22 enhance skin inflammation by stimulating the secretion of IL-1beta by keratinocytes via the ROS-NLRP3caspase-1 pathway[END_REF], suggesting that in the presence of several pro-inflammatory cytokines (IFN-γ, TNF-α, IL-17 and IL-22) a strong increase in pro-inflammatory caspases could be obtained. This hypothesis is in agreement with what happens in a psoriatic skin, where keratinocytes are in direct contact with a huge amount of pro-inflammatory cytokines secreted from the recruited immune cells and from themselves.

Our data showing that keratinocytes are primed to secrete IL-1β through the activation of inflammasome reinforces the idea that keratinocytes are immunological active cells [START_REF] Feldmeyer | Interleukin-1, inflammasomes and the skin[END_REF]. In fact, pro-IL-1β is constitutively synthetized in keratinocytes, making these cells the principal source of IL-1 in the skin [START_REF] Lee | Dynamic expression of epidermal caspase 8 simulates a wound healing response[END_REF] released under stress conditions. However, bone marrow transplantation experiment indicated that the activation of pro-inflammatory caspases in immune cells is sufficient to induce a fully inflammatory response in mice, suggesting that caspase-mediated IL-1β production and secretion by the recruited macrophages, neutrophils and other immune cells could be sufficient to develop the psoriasis-like phenotype. It is noteworthy that inflammasome components are not so well expressed in murine keratinocytes and accordingly, murine keratinocytes are not able to activate inflammasome in response to stimuli [START_REF] Sand | Expression of inflammasome proteins and inflammasome activation occurs in human, but not in murine keratinocytes[END_REF].

Therefore, although in our study we postulated that activation of pro-inflammatory caspases in immune cells, and more likely in macrophages, is sufficient to develop a psoriasis-like phenotype in mice, it appears that the case of humans the activation of inflammatory caspases in keratinocytes could be determinant in the development of the disease.

In conclusion, the findings presented herein confirm that pro-inflammatory caspases are involved in psoriasis pathogenesis, highlighting the notion that control of pro-inflammatory caspase activation and IL-β maturation/secretion is essential for the improvement of chronic inflammatory disease.

Materials and methods

Study approval.

This study was conducted on biopsies from 25 patients enrolled in a study registered in Mice. Mice were housed under specific pathogen-free conditions and fed with standard laboratory food. All mice (WT, C1/C11 deficient and Lyn∆N) were on a C57BL/6J genetic background and were used at 8-10 weeks of age, except for adoptive transfer, where mice were irradiated and reconstituted at six weeks. Both males and females were used in this study. WT mice (C57Bl6/J) were purchased from Envigo and C1/C11 deficient mice were a gift from Dr Richard A Flavell (Yale University, New Haven, CT, USA).

Figure legends

  clinicaltrials.gov under the identifier NCT01538342: Role of Tyrosine Kinase Lyn and Cleaved Form by Caspases in Psoriasis. This study was approved by the local ethics committee (Comité de Protection des Personnes Sud-Méditerranée V, n° 2011-A00786-35.065), and was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practices guidelines. All patients and healthy donors included signed informed consent before their inclusion in the study. The ethics review board from the French Minister for the High Education and Research approved all animal studies together with the Institutional Animal Care and Use Committee of the Centre Méditerranéen de Médecine Moléculaire (C3M), Nice, France (INSERM U1065).

Figure 1 :

 1 Figure 1: Pro-inflammatory caspases are activated in lesional skin biopsies from psoriasis patients. Protein extraction was performed from total skin of healthy donors (HD) and non-lesional (NL) and lesional (L) skin biopsies from psoriasis patients and then, western blot analysis were carried out. a, b and c: Caspase 1, 4 and 5 were blotted in HD and in NL and L biopsies from different psoriasis patients showing caspase activation in lesional samples. d: Total of analyzed patients showing in how many patients activated caspases are present. * Unspecific band.

Figure 2 :

 2 Figure 2: Crossing of Lyn∆N mice with C1/C11 deficient mice leads to a reduction in psoriasis-like phenotype. Lyn∆N mice were crossed with C1/C11 deficient mice (dKO mice). Newborn mice, Lyn∆N C1/C11 deficient mice (Lyn∆N dKO mice), were sacrificed, together with WT and Lyn∆N mice, 11 days after birth and abdomen skin was harvested. a: Adapted PASI score was assessed at three time points (see Materials and Methods), number of analyzed mice is showed below the graph. b: Survival of WT, Lyn∆N and Lyn∆N dKO through the eleven days of the study. c: Representative histological sections stained with haematoxylin and eosin (10X and 20X), scale bars = 50 µm. d: Epidermal hyperplasia quantification through haematoxylin and eosin staining, (WT, n = 5; Lyn∆N, n = 7; Lyn∆N dKO, n = 10). e: Skin was processed for total RNA isolation and gene expression was determined by qPCR (WT, n = 4; Lyn∆N, n = 4; Lyn∆N dKO, n = 8). f: Total protein was prepared and IL-1β (WT, n = 5; Lyn∆N, n = 8; Lyn∆N dKO, n = 14) and IL-18 (WT, n = 3; Lyn∆N, n = 5; Lyn∆N dKO, n = 7) levels were quantified by ELISA. Data presented were obtained from pooled mice of several experiments. A.U: arbitrary units. Errors bars represent mean ± SD. One-way ANOVA with the uncorrected Fisher's LSD multiple comparison test was applied to determine statistical significance in the PASI score. Long-rank test (Mantel-Cox test) was applied for survival experiment and Student t test was applied for the rest of panels, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns: non-significant.

Figure 3 :

 3 Figure 3: Deficiency in C1/C11 genes in mice decreases IMQ-induced psoriasis-like phenotype. WT and C1/C11 deficient mice (dKO mice) were treated daily for six days with vehicle (Vaseline (Vasel)) or imiquimod (IMQ) cream and after sacrificed them back skin was harvested. a: Severity of IMQ-induced psoriasis like phenotype represented by Erythema, Scaling and Cumulative score measurement, (Vasel, n = 2; IMQ-WT, n = 4; IMQ-dKO, n = 8). b: Representative histological sections stained with haematoxylin and eosin (10X and 20X), scale bars = 50 µm. c: Epidermal hyperplasia quantification through haematoxylin and eosin staining, (Vasel, n = 3; IMQ-WT, n = 4; IMQ-dKO, n = 8). d: The number of immune cell populations was quantified by FACS, (Vasel, n = 3; IMQ-WT, n = 4; IMQ-dKO, n = 8). e: Skin was processed for total RNA isolation and gene expression was determined by qPCR (Vasel, n = 3; IMQ-WT, n = 4; IMQ-dKO, n = 8). f: Skin was processed for total protein extraction and IL-1β and IL-18 levels were quantified by ELISA, (Vasel, n = 3; IMQ-WT, n = 4; IMQ-dKO, n = 8). Data represent one experiment representative of two independent experiments. A.U: arbitrary units. Errors bars represent mean ± SEM. Student t test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ns: non-significant.

Figure 4 :

 4 Figure 4: Inhibition of Caspase-1 by AC-YVAD-CMK compound reduces IMQ-induced psoriasis-like phenotype in mice. WT mice were treated daily for six days with vehicle (Vaseline (Vasel)) or imiquimod (IMQ) cream in combination with dimethylsulfoxide (DMSO) or the caspase-1 inhibitor AC-YVAD-CMK (8 mg/kg). a: Severity of IMQ-induced psoriasis like phenotype represented by Erythema, Scaling and Cumulative score measurement. b: Representative histological sections stained with haematoxylin and eosin (10X), scale bars = 50 µm. c: Epidermal hyperplasia quantification through haematoxylin and eosin staining. d: Skin was processed for total RNA isolation and gene expression was determined by qPCR. e: Skin was processed for total protein extraction and IL-1β and IL-18 levels were quantified by ELISA. A.U: arbitrary units. Errors bars represent mean ± SEM. Student t test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns: non-significant. (a,d : Vasel/DMSO, n = 6; Vasel/YVAD, n=2; IMQ/DMSO, n = 7; IMQ/YVAD, n=11 pooled mice from two independent experiments; c,e : data represent one experiment representative of two independent experiments).

Figure 5 :

 5 Figure 5: Primary human keratinocytes are primed to secrete IL-1β and activate caspase 5 in mimicking psoriasis conditions. Primary human keratinocytes were cultured in KGM and then stimulated with the proinflammatory cytokines TNF-α (10 ng/ml) and IL-17A (200 ng/ml) at different time points and after total RNA isolation, caspase 1, caspase 4, caspase 5 (a); IL-1β (b); and NLRP-1 and NLRP-3 (c) gene expression was determined by qPCR, (n = 3 independent experiments). d, e, and f: Total lysates were prepared from stimulated keratinocytes and Western Blot analyses were performed, (n = 3 independent experiments). g: Primary human keratinocytes were stimulated with TNF-α (10 ng/ml) and IL-17A (200 ng/ml) in the presence or not of nigericin to activate inflammasome, then IL-1β and caspase 1 expression was determined intracellular and extracellularly, (n = 3 independent experiments). h: After keratinocytes stimulation with TNF-α (10 ng/ml) and IL-17A (200 ng/ml) in the presence of nigericin at two time points, supernatant was harvested and IL-1β secretion was quantified by ELISA, (n = 3 independent experiments). A.U: arbitrary units. Errors bars represent mean ± SEM. Student t test, * p ≤ 0.05, ** p ≤ 0.01, ***, p ≤ 0.001, **** p ≤ 0.0001, ns: non-significant. T: TNF-α (10 ng/ml), I: IL-17A (200 ng/ml).

Figure 6 :

 6 Figure 6: Caspase 1/11 deficiency in immune cells is enough to reduce IMQ-induced psoriasis like phenotype. WT and C1/C11 deficient (dKO) mice were irradiated and then reconstituted with bone marrow from WT (WT→WT and WT→dKO) and dKO (dKO→dKO and dKO→WT) mice, which were left to recuperate for 8 weeks. Then, the four groups of mice were treated daily for six days with vehicle (Vaseline (Vasel)) or imiquimod (IMQ) cream. After treatment back skin was harvested. a: Severity of IMQ-induced psoriasis like phenotype represented by Erythema, Scaling and Cumulative score measurement. b: Representative histological sections stained with haematoxylin and eosin (10X), scale bars = 50 µm. c: Epidermal hyperplasia quantification through haematoxylin and eosin staining, (for each group of mice, Vasel, n = 3; IMQ, n = 6). d: Skin was processed for total RNA isolation and gene expression was determined by qPCR. e: Skin was processed for total protein extraction and IL-1β levels were quantified by ELISA, (for each group of mice, Vasel, n = 3; IMQ, n = 6).

Acknowledgments

This work was supported by grants from the Fondation pour la Recherche Médicale (DMP20101120387), from the Fondation ARC pour la Recherche sur le Cancer (PGA1RF201702053889), the INSERM and the University of Nice-Sophia-Antipolis. This work was supported by a grant from the Clinical Research Hospital Program (PHRC 2011) from the French Ministry of Health. This work and LEA were funded by the French Government (National Research Agency, ANR) through the 'Investments for the Future' LABEX SIGNALIFE: program reference #ANR-11-LABX-0028-01. We thank Dr Abdallah Khemis (Dermatology department, CHU Nice), Pr Marie-Aleth Richard (Dermatology department, CHU Marseille) and Pr Didier Bessis (Dermatology department, CHU Montpellier) for Patient recruitment and skin biopsies. We thank Pr Richard A. Flavell for providing C1/C11 deficient mice. We thank Dr Karine Bille and Dr Corine Bertolotto for provide us the Normal Human Keratinocytes. We thank the Animal facility and the Imaging Facility of C3M. We thank la Délégation à la Recherche Clinique et à l'Innovation for their help in the PHRC. We thank le Conseil Général des AM et de la region PACA et Corse for their help.

Conflict of interest

There is no conflict of interest declared by the authors.

Author contributions

LEA and DG obtained the majority of data presented herein and were assisted by JPB, CRB, JC, LM, MG, PLO and PC. JPO, JPL and TP included patients and performed clinical examination. SM and LEA wrote and edit the manuscript. JER and PA provided intellectual input and edit the paper. SM conceived the project, supervised the study and obtained funding. All authors reviewed and approved the final version of the manuscript prior to its submission for publication.


Géraldine Albérola

Jens-Michael Schröder

Carine Froment

Michel Simon
email: michel.simon@inserm.fr

The Amino-Terminal Part of Human Filaggrin-2 is a Component of Cornified Envelopes

Keywords: corneocyte, epidermis, terminal differentiation, keratinocyte, transglutaminase, mass spectrometry CE, cornified envelope, FLG2, filaggrin-2, TGase, transglutaminase

2

TO THE EDITOR

Filaggrin-2 (FLG2), a member of the S100-fused type protein family, is a 250 kDa protein mainly expressed in differentiated keratinocytes of the epidermis [START_REF] Wu | Molecular identification and expression analysis of filaggrin-2, a member of the S100 fused-type protein family[END_REF][START_REF] Hsu | Deimination of human filaggrin-2 promotes its proteolysis by calpain 1[END_REF]. Its expression is reduced in atopic dermatitis [START_REF] Pellerin | Defects of filaggrin-like proteins in both lesional and nonlesional atopic skin[END_REF], and FLG2 gene heterozygous non-sense mutations are associated with a more persistent form of the disease in Afro-American populations [START_REF] Margolis | Filaggrin-2 variation is associated with more persistent atopic dermatitis in African American subjects[END_REF]. Loss of FLG2 due to homozygous mutations is responsible for peeling skin syndrome type A [START_REF] Bolling | Generalized Ichthyotic Peeling Skin Syndrome due to FLG2 Mutations[END_REF][START_REF] Mohamad | Filaggrin 2 Deficiency Results in Abnormal Cell-Cell Adhesion in the Cornified Cell Layers and Causes Peeling Skin Syndrome Type A[END_REF]. FLG2 consists of an S100-homologous NH2-terminal domain, a spacer domain, a large central domain and a short COOH-tail (Figure 1a). The central domain is formed by two types of 75-77 amino-acids repeats: nine A-type repeats and 14 B-type repeats. During cornification FLG2 is proteolytically processed, the various domains being released. The COOH-tail exhibits anti-microbial activity [START_REF] Hansmann | Skin-Derived C-Terminal Filaggrin-2 Fragments Are Pseudomonas aeruginosa-Directed Antimicrobials Targeting Bacterial Replication[END_REF]. The B-type repeats are first located in the matrix of the lower corneocytes and degraded later [START_REF] Hsu | Deimination of human filaggrin-2 promotes its proteolysis by calpain 1[END_REF]. The resulting amino-acids are suspected to be part of the natural moisturizing factor. The role of the amino-terminal part, including the A-type repeats, is not known. Here, we demonstrate that it is cross-linked to the cornified envelope (CE).

CEs were purified, as previously reported [START_REF] Simon | Evidence that filaggrin is a component of cornified cell envelopes in human plantar epidermis[END_REF], from plantar stratum corneum obtained from two patients undergoing podiatry care but who had no history of skin diseases and had given their written informed consent. The CEs were then digested with endoproteinase V8. The resulting polypeptides were separated in SDS-gel, in-gel digested with trypsin, and identified using Nanoflow liquid chromatography-tandem mass spectrometry (Supplementary Materials online). One hundred and twenty-six proteins, with various types and cellular location, were identified. Some of them are well known CE components, including keratins, filaggrin, hornerin, desmosomal proteins, cystatin-alpha and transglutaminases (TGases) 1, 3 and 5 (Figure 1b-d and Supplementary Table S1. The complete list is also available upon request to MS). Eleven unique peptides derived from FLG2 were also identified (Figure 1; Supplementary Table S2). They mostly corresponded to the S100, the spacer and the type-A repeat domains (3 peptides in each domain). In agreement with this result, two peptides corresponding to the spacer domain have been previously shown to be incorporated in CEs [START_REF] Vermeij | ROS quenching potential of the epidermal cornified cell envelope[END_REF]. However, peptides derived from hornerin, a known component of CEs [START_REF] Henry | Hornerin is a component of the epidermal cornified cell envelopes[END_REF][START_REF] Hsu | Deimination of Human Hornerin Enhances its Processing by Calpain-1 and its Cross-Linking by Transglutaminases[END_REF], were distributed all along the protein sequence (Supplementary Table S3). Further supporting the hypothesis that FLG2 Atype repeats are cross-linked to CEs, are their sequence homology with hornerin (50-75% identity), and their high content in glutamine which may act as amine acceptor in the formation of NƐ(γ-glutamyl)-lysyl isopeptide bonds catalysed by TGases.

To confirm that the amino-terminal part of FLG2 is a component of CEs, the plantar envelopes (with two additional samples) and CEs purified from abdominal skin were stained with an antibody directed against the spacer domain [START_REF] Wu | Molecular identification and expression analysis of filaggrin-2, a member of the S100 fused-type protein family[END_REF]. Abdominal skin samples, from three females without any history of skin diseases undergoing plastic surgery, was obtained from Genoskin (Toulouse, France), following written informed consent of the donors and as agreed by the French Ministry of Research (#AC-2017-2897). Using indirect immunofluorescence, the CEs were clearly labelled (Figure 2a). Roughly one out of two CEs was labelled, whatever the anatomic territory (see Supplementary Table S4). The purified CEs were then incubated for increasing periods of time with endoproteinase V8, as previously reported [START_REF] Hsu | Deimination of Human Hornerin Enhances its Processing by Calpain-1 and its Cross-Linking by Transglutaminases[END_REF], and the fragments produced were analyzed by immunoblotting (Figure 2b). As typically observed when CEs are proteolysed, multiple high molecular weight bands with a smearing pattern (that differed between plantar and abdominal CEs) were immunostained after the protease action.

We tested whether the FLG2 spacer domain was a substrate of TGases, as previously described [START_REF] Hsu | Deimination of Human Hornerin Enhances its Processing by Calpain-1 and its Cross-Linking by Transglutaminases[END_REF]. A recombinant form of the spacer (FLG2 residues 96 to 235; Figure 2c) was incubated with TGase-1 or TGase-3, in the presence of monodansylcadaverine.

Samples were then separated by SDS-gel electrophoresis and gels observed under UV light (Figure 2d). TGase-1 and TGase-3 induced a time-dependent incorporation of monodansylcadaverine in the spacer and in high-molecular-weight cross-linked complexes (indicated by dots).

Finally, we showed that reduced amount of FLG2 induced a lower mechanical resistance of CEs purified from reconstructed human epidermis (Figure 2e).

In conclusion, our data demonstrate that FLG2 N-terminal part, including A-type repeats, is incorporated in CEs. Thus FLG2 and filaggrin have overlapping roles in the homeostasis of the epidermal barrier. Three non-exclusive possibilities may explain the stratum corneum fragility observed in the absence of FLG2, as in peeling skin syndrome type A patient skin: 1) an abnormal intermediate filament compaction, since mouse Flg2 has been shown to interact with and aggregate keratin filaments in vitro (Presland et al, 2006); 2) a reduced corneocyte resistance, as suggested by our data; 3) an induced defect in keratinocyte differentiation program, as seen in the patient skin and in reconstructed human epidermis [START_REF] Pendaries | In a threedimensional reconstructed human epidermis filaggrin-2 is essential for proper cornification[END_REF]. The CEs were analyzed by immunoblotting with the same antibody after incubations without (V8-) or with (V8+) V8 protease for 0 to72 h, as indicated. (c) The recombinant filaggrin-2 spacer domain (rFLG2) was Coomassie blue stained after separation by SDS-gel electrophoresis. Although its theoretical molecular mass is 15.66 kDa, two bands around 27 and 55 kDa were observed. (d) Casein, as a control, and rFLG2 were incubated with either transglutaminase 1 (TGase-1) or transglutaminase 3 (TGase-3) for 0, 1 and 4 hours in the presence of CaCl 2 and monodansylcadaverine. Samples were separated by SDS-gel electrophoresis, and incorporation of monodansylcadaverine was revealed by UV illumination.

•, high-molecular-weight cross-linked complexes. The position of molecular mass markers (m) is indicated on the right. (e) Reconstructed human epidermis were produced with keratinocytes from one donor and transduced with either control shRNA (shCo) or FLG2specific shRNA (shFLG2). In each condition, CEs were purified from four pooled epidermis.

The same amount of CEs were then sonicated for 5 seconds (n = 6 independent experiments), and the remained intact envelopes were observed under a dark field microscope and quantified.

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Filaggrin-2 spacer domain is cross-linked to cornified envelopes. (a) Cornified

ACKNOWLEDGMENTS

We are indebted to Dr Pascal Descargues (Genoskin, Toulouse, France) for the human skin samples, to Mikaël Pancarte for advices in envelope sonication, and to the technical assistance of Carole Pons. This work was supported by INSERM, Toulouse III University, and French Society of Dermatology. We thank Odile Burlet-Schiltz for access to the Proteome Infrastructure of Toulouse. This work was supported, in part by the Région Occitanie, European funds (Fonds Européens de Développement Régional, FEDER), Toulouse Métropole, and by the French Ministry of Research with the Investissement d'Avenir Infrastructures Nationales en Biologie et Santé program (ProFI, Proteomics French Infrastructure project, ANR-10-INBS-08). ORCID Michel Simon: http://orcid.org/0000-0003-3655-6329

CONFLICT OF INTEREST

The authors state no conflict of interest.