Rosario Brunetto
email: rosario.brunetto@ias.u-psud.fr

C Lantz

T Nakamura

D Baklouti

T Le Pivert-Jolivet

S Kobayashi

F Borondics

Characterizing irradiated surfaces using IR spectroscopy

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

Introduction

The remote sensing observations of Solar System airless bodies deal with the problem of the surfaces being constantly modified by exogenous alterations. For bodies in the inner Solar System, slow solar wind ion irradiation and micrometeoroid impacts are driving the alteration of airless surfaces, with solar wind dominating the alteration across most surface types [START_REF] Brunetto | Asteroid Surface Alteration by Space Weathering Processes[END_REF]. Even if slow solar wind ions penetrate the surface less than 1 µm, they significantly affect the optical properties of the upper layers [START_REF] Noguchi | Space Weathered Rims Found on the Surfaces of the Itokawa Dust Particles[END_REF][START_REF] Bonal | Visible-IR and Raman Microspectroscopic Investigation of Three Itokawa Particles Collected by Hayabusa: Mineralogy and Degree of Space Weathering Based on Nondestructive Analyses[END_REF][START_REF] Loeffler | Irradiation of Olivine by 4 keV He + : Simulation of Space Weathering by the Solar Wind[END_REF] which are the ones probed by the remote sensing observations. Microstructural/chemical modifications by solar wind irradiation may include the production of disordered rims (amorphous or partly amorphous), implantation vesicles, solar flare tracks, nanophase-Fe-bearing particles, which all contribute to some extent to the alteration of surface spectral properties. As we induce the composition of the bulk of the object from the observation of the upper layers, the irradiation may seriously affect the spectral identification of underlying pristine materials. The resulting biases hamper both the 2 understanding of the spectral diversity of small bodies and their link to extraterrestrial materials available in our laboratories.

The ion irradiation of surface regolith is known to be particularly intense on the Moon [START_REF] Hapke | Space Weathering from Mercury to the Asteroid Belt[END_REF]. However, asteroid surfaces are on average much younger than the Moon's "undisturbed" terrains and their regolith has a much higher mobility due to the low gravity. So it was remarkable to observe that even young asteroid families show clear effects of surface aging [START_REF] Vernazza | Solar Wind as the Origin of Rapid Reddening of Asteroid Surfaces[END_REF]). As a matter of fact, the solar wind fluxes in the inner Solar System are so intense that the astrophysical time necessary to produce any modification of the asteroid surfaces is lower than 1 Myr [START_REF] Strazzulla | Spectral Alteration of the Meteorite Epinal (H5) Induced by Heavy Ion Irradiation: A Simulation of Space Weathering Effects on near-Earth Asteroids[END_REF][START_REF] Hapke | Space Weathering from Mercury to the Asteroid Belt[END_REF]). This was confirmed by the laboratory analysis of samples collected from the young surface of the asteroid Itokawa by the Hayabusa mission. The Itokawa grains showed incipient space weathering effects [START_REF] Noguchi | Incipient Space Weathering Observed on the Surface of Itokawa Dust Particles[END_REF], with the presence of disordered rims resulting from solar wind radiation damage [START_REF] Noguchi | Space Weathered Rims Found on the Surfaces of the Itokawa Dust Particles[END_REF][START_REF] Keller | A Transmission Electron Microscope Study of Itokawa Regolith Grains[END_REF], and with the presence of solar flare tracks. Exposure ages estimated through the analysis of the solar flare track densities (calibrated for 1AU using lunar samples) were in the range 10 3 -10 6 years, depending on the considered particle [START_REF] Noguchi | Space Weathered Rims Found on the Surfaces of the Itokawa Dust Particles[END_REF][START_REF] Nagao | Irradiation History of Itokawa Regolith Material Deduced from Noble Gases in the Hayabusa Samples[END_REF].

Despite being a complicated source of spectral biases, surface weathering could provide a convenient tool to estimate the age of a young surface. The direct measurements of the exposure time of surface materials is currently only possible thanks to the laboratory analysis of samples collected and returned from space [START_REF] Nagao | Irradiation History of Itokawa Regolith Material Deduced from Noble Gases in the Hayabusa Samples[END_REF]) (e.g., the unique cases of Itokawa and the Moon, and soon asteroids Ryugu and Bennu). The remote sensing spectroscopy of irradiated surfaces can provide an additional aging chronometer, independent from the timescales derived from dynamical studies [START_REF] Brunetto | Modeling Asteroid Surfaces from Observations and Irradiation Experiments: The Case of 832 Karin[END_REF]). We thus have two good reasons to study how space weathering modifies the reflectance spectra of airless surfaces: to remove the biases affecting remote sensing and to estimate the age of the surface. However, it is mandatory to first establish reliable spectral proxies for distinguishing an irradiated from a pristine surface. These criteria must be based on unambiguous and reproducible spectral variations upon irradiation.

In the case of bright objects, the irradiation of exposed surfaces has mainly been studied by detecting the spectral variations induced by weathering agents (energetic ions, micrometeorites) in the visible and near-IR range (VIS-NIR, 0.4-2.5 µm), both through direct observations [START_REF] Binzel | Observed Spectral Properties of near-Earth Objects: Results for Population Distribution, Source Regions, and Space Weathering Processes[END_REF]) and simulation experiments [START_REF] Sasaki | Production of Iron Nanoparticles by Laser Irradiation in a Simulation of Lunar-like Space Weathering[END_REF]. Typical spectral variations included changes in the spectral slopes and reflectance [START_REF] Marchi | Space Weathering of near-Earth and Main Belt Silicate-Rich Asteroids: Observations and Ion Irradiation Experiments[END_REF]). These variations have been largely documented since the '70s in the case of the Moon [START_REF] Pieters | Optical Effects of Space Weathering: The Role of the Finest Fraction[END_REF], and since the '90s for bright asteroids [START_REF] Binzel | Observed Spectral Properties of near-Earth Objects: Results for Population Distribution, Source Regions, and Space Weathering Processes[END_REF][START_REF] Marchi | Space Weathering of near-Earth and Main Belt Silicate-Rich Asteroids: Observations and Ion Irradiation Experiments[END_REF]. For S-type asteroids, the reddening of the VIS-NIR spectral slope proved to be a good proxy of space weathering.

Unlike the case of bright objects such as S-type asteroids, a clear and univocal VIS-NIR weathering trend cannot be established in the case of the more numerous dark asteroids [START_REF] Nesvorný | Evidence for Asteroid Space Weathering from the Sloan Digital Sky Survey[END_REF][START_REF] Lazzarin | Space Weathering in the Main Asteroid Belt: The Big Picture[END_REF]. In previous work, we found that dark carbon-rich meteorites of various compositions showed different VIS-NIR spectral trends after ion irradiation [START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]). In addition, the VIS-NIR spectral slopes and albedo are known to be influenced by several factors other than composition, such as surface porosity, grain size and roughness. In the case of dark objects, these parameters induce VIS-NIR spectral variations that in some cases are comparable to or larger than the weathering trends [START_REF] Lantz | Ion Irradiation of the Murchison Meteorite: Visible to Mid-Infrared Spectroscopic Results[END_REF]. For all these reasons, an unambiguous spectral weathering trend for primitive objects cannot be determined using VIS-NIR data alone [START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]). Additional information is required from other spectral ranges with diagnostic bands, such as the mid-IR (MIR, 2.5-17 µm) and far-IR (FIR, 17-100 µm) ranges.

In previous studies [START_REF] Lantz | Ion Irradiation of the Murchison Meteorite: Visible to Mid-Infrared Spectroscopic Results[END_REF][START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF][START_REF] Brunetto | Ion Irradiation of Allende Meteorite Probed by Visible, IR, and Raman Spectroscopies[END_REF], we reproduced the solar wind weathering of primitive asteroids in the laboratory, by irradiating a number of carbonaceous chondrites from different sub-classes (including CM Murchison and Mighei, CO Lancé and Frontier Mountain 95002, CI Alais, CV Allende, and C2 Tagish Lake) with He + ions. We studied the laboratory-irradiated samples using reflectance spectroscopy and more recently micro-spectral imaging [START_REF] Brunetto | Hyperspectral FTIR Imaging of Irradiated Carbonaceous Meteorites[END_REF]. Ion irradiation of the meteorites induced significant IR spectral variations. A comparison of the spectral imaging data at different wavelengths demonstrates that IR photons from the center of the reststrahlen bands (surface scattering) are significantly more efficient in detecting irradiated areas on the sample than visible photons and IR photons in volume scattering ranges (see Appendix A and Fig. A1).

To explore to what extent the IR bands are a good proxy of irradiation, we decided to analyze our set of meteorite samples with FIR spectroscopy (in almost all our previous analyses the measured spectral range stopped at 15 μm [START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]. In this paper we report the results of these new measurements, plus a new irradiation experiment performed on a polished section of the CM2 Murchison meteorite.

Experimental setup

Laboratory irradiation of meteorites

The preparation of the pellets (13 mm diameter) of the CV3 Allende, CO3.5 Lancé, CO3 FM95002, CM2 Murchison, CM2 Mighei, CI1 Alais, and C2-ung Tagish Lake meteorites is reported elsewhere [START_REF] Brunetto | Ion Irradiation of Allende Meteorite Probed by Visible, IR, and Raman Spectroscopies[END_REF][START_REF] Lantz | Ion Irradiation of the Murchison Meteorite: Visible to Mid-Infrared Spectroscopic Results[END_REF][START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]. A new 1-cm sized chip of the CM2 Murchison meteorite with a flat surface with roughness made by a #400 polishing disk was prepared at Tohoku University in Japan. All the samples were irradiated at the SIDONIE implanter of CSNSM-Orsay (France), using He + ion beams with 40 keV energy (20 keV for the Murchison chip), at ion flux of ~10 13 ions/(sec cm 2 ) up to a total fluence of ~6x10 16 ions/cm 2 . The samples were monitored in situ under vacuum by VIS-NIR diffuse reflectance spectroscopy. Details of the irradiation conditions are described in our previous studies [START_REF] Brunetto | Ion Irradiation of Allende Meteorite Probed by Visible, IR, and Raman Spectroscopies[END_REF][START_REF] Lantz | Ion Irradiation of the Murchison Meteorite: Visible to Mid-Infrared Spectroscopic Results[END_REF][START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF].

IR spectroscopy

The FTIR hyperspectral imaging measurements reported in Fig. A1 were acquired with an Agilent Cary 670/620 micro-spectrometer equipped with a 128x128 pixel Focal Plane Array detector, installed at the SMIS beamline of the SOLEIL synchrotron (France). Mid-IR spectra were measured using the internal globar source at 8 cm -1 spectral resolution. The infrared maps were acquired in reflectance geometry, with respect to gold references. More details can be found elsewhere [START_REF] Brunetto | Hyperspectral FTIR Imaging of Irradiated Carbonaceous Meteorites[END_REF]. The MIR part of the spectra reported in Section 3 was measured in a previous study [START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]) using a spectral resolution of 4 cm -1 .

The new FIR spectra reported in this study (Figs. 123) were acquired at the SMIS beamline using a NicPlan microscope, coupled to a iS50 FTIR spectrometer (Thermo Fisher) operating in confocal reflection geometry, with respect to gold references. To measure in the far-IR range (700-100 cm -1 or 14-100 µm), we used a bolometer detector (boron doped silicon, 4.2 K cooled, Infrared Laboratories) and a solid-state Si beamsplitter. The spectral resolution was 4 cm -1 (constant in wavenumber, but variable in wavelength, typically ~0.16 µm in the 20 µm region and ~0.98 µm in the 50 µm region) and the spectral sampling 0.5 cm -1 (typically ~0.02 µm in the 20 µm region and ~0.125 µm in the 50 µm region). We analyzed irradiated and unirradiated areas on each sample, using spot sizes of about 300 µm in diameter per spectrum. The various spectra (typically 5 spectra per meteorite) of the unirradiated spots were averaged, as well as the irradiated spots, to finally produce average spectra representative of the unirradiated and irradiated meteorites. Thus, we averaged the small scale heterogeneity of the chosen chondrites. The bolometer also allowed us to acquire data in the 750-1050 cm -1 (13.3-9.52 µm) range. Although noisy, this additional spectral range proved to be extremely useful to properly combine spectral ranges obtained with different detectors (see Fig. 2). The FIR spectra were scaled to match the MIR spectra in the overlapping region. The combined MIR-FIR spectra were then baseline corrected using a convex hull. The baseline correction was performed to focus on the position and shape of the IR spectral bands, and to allow comparison with IR remote sensing data of asteroids whose continuum is usually taken into account and removed in the thermal model of the spectral energy distribution [START_REF] Emery | Thermal Emission Spectroscopy (5.2-38 μm) of Three Trojan Asteroids with the Spitzer Space Telescope: Detection of Fine-Grained Silicates[END_REF]. We calculated the peak positions of the IR bands from the 1st derivative of the spectra (Table 1) and the error is estimated based on the spectral sampling and the noise in the data (increasing with decreasing wavenumber). A similar procedure to determine the peak positions was applied to remote sensing data of Bennu and Hektor asteroids (see Section 4) and an additional double-check was obtained from polynomial fits around the center of the bands.

In our microscopes we used Schwarzschild objectives, which accept photons in a large range of angles at all azimuths. The geometry of the spectral measurements can influence the spectral shape (Salisbury et al. 1991;[START_REF] Salisbury | Mid-Infrared Spectroscopy: Laboratory Data[END_REF], thus a direct comparison between reflectance and emissivity spectra must be taken with great care. In this paper we do not try to necessarily find the best laboratory match to asteroid spectral data, but we rather focus on relevant modifications of spectral features induced by ion irradiation.

Irradiation experiments and IR spectral measurements were performed in two different laboratories, and the spectra were measured ex situ in laboratory air. Potential air-exposure effects such as oxidation cannot be excluded. However, if present, they do not seem to have any significant effect over time on the spectral modifications shown here, as we have verified by comparing the FIR spectra obtained on Allende in the new measurements with one Allende FIR spectral measurement reported in our previous work [START_REF] Brunetto | Ion Irradiation of Allende Meteorite Probed by Visible, IR, and Raman Spectroscopies[END_REF]).

Results

FIR spectral effects of He implantation in carbonaceous chondrites

We report in Fig. 1 the new laboratory spectra of virgin and irradiated meteorites measured in the FIR range. In Fig. 2 we illustrate the combination of FIR and MIR ranges, as described in Section 2.2, with the examples of a meteorite whose matrix is rich in hydrated minerals (C2-ung Tagish Lake) and a meteorite whose matrix is rich in anhydrous phases (CO3 FM95002). The full collection of combined MIR-FIR spectra will be available at the DAYSY spectral database hosted by the SSHADE [START_REF] Schmitt | SSHADE: The European Solid Spectroscopy Database Infrastructure[END_REF]) Solid Spectroscopy database infrastructure (www.sshade.eu).

Different classes of carbonaceous chondrites have different IR spectra (Salisbury et al. 1991;[START_REF] Beck | Transmission Infrared Spectra (2-25μm) of Carbonaceous Chondrites (CI, CM, CV-CK, CR, C2 Ungrouped): Mineralogy, Water, and Asteroidal Processes[END_REF][START_REF] Vernazza | DIFFERENT ORIGINS OR DIFFERENT EVOLUTIONS? DECODING THE SPECTRAL DIVERSITY AMONG C-TYPE ASTEROIDS[END_REF][START_REF] Hamilton | Spectral Classification of Ungrouped Carbonaceous Chondrites II: Parameters and Comparison to Independent Measures[END_REF][START_REF] Hamilton | Evidence for Widespread Hydrated Minerals on Asteroid (101955) Bennu[END_REF]. Just like the MIR range is characterized by the strong Si-O stretching bands in silicates, the FIR range is dominated by the bending vibrations of the Si-O bonds and translation modes of metal ions [START_REF] Jäger | Steps toward Interstellar Silicate Mineralogy. IV. The Crystalline Revolution[END_REF]. Both MIR and FIR are thus very sensitive to the composition of the silicates and the thermal history of the meteorites [START_REF] Beck | Transmission Infrared Spectra (2-25μm) of Carbonaceous Chondrites (CI, CM, CV-CK, CR, C2 Ungrouped): Mineralogy, Water, and Asteroidal Processes[END_REF]. Our analysis confirms that the FIR spectra of meteorites whose matrix is rich in anhydrous phases (CV3 Allende, CO3.5 Lancé, CO3 FM95002) and those that are rich in aqueously altered phases (CM2 Murchison, CM2 Mighei, CI1 Alais, and C2-ung Tagish Lake) are significantly different.

Significant differences are also observed when comparing spectra before and after He + irradiation. The spectra of Frontier Mountain 95002 and Lancé are very similar and they both show typical olivine bands corresponding to a composition of Fo~50-60, estimated based on literature data of olivine [START_REF] Hamilton | Thermal Infrared (vibrational) Spectroscopy of Mg-Fe Olivines: A Review and Applications to Determining the Composition of Planetary Surfaces[END_REF]. After irradiation, the bands are shifted to peak positions that are observed in olivines with Fo~30-35. Similar Fo values are found based on the MIR bands, both before and after irradiation [START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]. The spectra of Alais and Tagish Lake share some similarities in the 450-500 cm -1 (20-22.2 µm) region, but they differ significantly for wavenumbers below 400 cm -1 (above 25 µm), both before and after irradiation. Finally, the spectra of Murchison and Mighei also share many similarities (both are CM chondrites). Overall, almost all bands are modified by irradiation, either in terms of shape/intensity and/or in terms of peak position.

It is particularly interesting to observe how the relative intensity of the doublets observed at about 500 cm -1 (20 µm) and 400 cm -1 (25 µm) in CO/CV and at about 450 cm -1 (22.2 µm) in CI/C2 are modified by irradiation. Upon irradiation, the left band of these doublets is always reduced in intensity with respect to the right band. It is obvious on the 450 cm -1 (22.2 µm) doublet (typical of saponite [START_REF] Beck | Transmission Infrared Spectra (2-25μm) of Carbonaceous Chondrites (CI, CM, CV-CK, CR, C2 Ungrouped): Mineralogy, Water, and Asteroidal Processes[END_REF])) of Alais and Tagish Lake, and on the low wavenumber bands of FM95002 and Lancé whose relative intensities change in a very similar way after irradiation. The ratio in relative intensity (I_left band / I-right band) before irradiation is about 1.47 for FM95002, 1.69 for Lancé, 1.01 for Tagish Lake, and 1.02 for Alais. After irradiation the ratio is about 1.09 for FM95002, 1.08 for Lancé, 0.91 for Tagish Lake, and 0.94 for Alais. The relative reduction upon irradiation is thus about 30-40% for FM95002 and Lancé and about 10% for Tagish Lake and Alais. It is interesting to observe that for almost all the considered meteorites the band near 350 cm -1 (28.6 µm) also increases in relative strength compared to bands at higher wavenumbers. Overall, the reflectance at lower wavenumbers is increasing with irradiation (see also the case of Allende in Fig. 3), and this can be due both to an increase of spectral slope (similarly to what is observed in the visible range for S-types) and to a modification of the relative intensity between lower and higher wavenumber bands. However, considering that the spectra have been baseline corrected, we attribute the observed effect to a change in relative band intensity. In particular, from the non-normalized spectra of Fig. 2, we can clearly observe a reduction of the intensity (and partly a broadening) of the bands in the higher wavenumbers, a phenomenon which seems to be much weaker for bands at lower wavenumbers. Fig. 1. FIR spectra of six meteorite samples before (black curves) and after (grey) irradiation. Frontier Mountain 95002, Lancé, Tagish Lake, Alais, and Mighei were irradiated with 6x10 16 He + /cm 2 at 40 keV. Murchison spectra were acquired on a polished chip sample irradiated with 6x10 16 He + /cm 2 at 20 keV. Vertical lines are drawn to guide the eye and stress the irradiation effect. The peak positions are reported in Table 1 andused [START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]) and hydrous [START_REF] Nakamura | Mineralogy and Noble-Gas Signatures of the Carbonate-Rich Lithology of the Tagish Lake Carbonaceous Chondrite: Evidence for an Accretionary Breccia[END_REF]) chondrites, respectively. The 830-1100 cm -1 (9-12 µm) and the 200-500 cm -1 (20-50 µm) ranges are characterized by the presence of Si-O stretching and bending vibration bands respectively. In the case of FM 95002, the bands are mainly due to anhydrous minerals (in particular olivine [START_REF] Hamilton | Thermal Infrared (vibrational) Spectroscopy of Mg-Fe Olivines: A Review and Applications to Determining the Composition of Planetary Surfaces[END_REF]). In the case of Tagish Lake, most bands can be attributed to clay minerals (in particular saponite [START_REF] Beck | Transmission Infrared Spectra (2-25μm) of Carbonaceous Chondrites (CI, CM, CV-CK, CR, C2 Ungrouped): Mineralogy, Water, and Asteroidal Processes[END_REF]). In this surface scattering regime, the peaks point towards the top. Vertical lines have been drawn to guide the eye along the position of the main reststrahlen bands and to stress the irradiation effect. The MIR range (1200-650 cm -1 ) has been measured in a previous study [START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]).

The silicate FIR bands are observed to shift in peak position upon irradiation. The irradiationinduced spectral shift is always observed towards the longer wavelengths (shorter wavenumbers, see Table 1), coherently with the MIR spectral shifts of the main silicate 10 µm feature observed in previous experiments on similar chondrites and on terrestrial silicates [START_REF] Vernazza | Paucity of Tagish Lake-like Parent Bodies in the Asteroid Belt and among Jupiter Trojans[END_REF][START_REF] Brunetto | Ion Irradiation of Allende Meteorite Probed by Visible, IR, and Raman Spectroscopies[END_REF][START_REF] Lantz | Ion Irradiation of the Murchison Meteorite: Visible to Mid-Infrared Spectroscopic Results[END_REF][START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]. Generally speaking, the stronger the band, the larger the shift, as already observed for irradiated Allende [START_REF] Brunetto | Ion Irradiation of Allende Meteorite Probed by Visible, IR, and Raman Spectroscopies[END_REF]. This is observed for all meteorites except Murchison. The shifts induced on CV and CO meteorites are very similar to each other. Also C2 Tagish Lake and CI Alais present very similar shifts after irradiation. Previous ion irradiation experiments on Tagish Lake using 1x10 16 He + /cm 2 at 200 keV reported a band shift of about 0.3 µm for the 9.8 µm (1020 cm -1 ) band and about 0.15 µm for the 22.3 µm (448 cm -1 ) band [START_REF] Vernazza | Paucity of Tagish Lake-like Parent Bodies in the Asteroid Belt and among Jupiter Trojans[END_REF], while in our case (6x10 16 He + /cm 2 at 40 keV) the shifts are about 0.4 µm (40 cm - 1 ) and 0.2 µm (4 cm -1 ) respectively, which could be explained by the fact that we are using a 6 times higher fluence (although at 5 times lower energy). The two CMs considered in this study behave differently from each other in terms of the shift of the main band at about 445-448 cm -1 (22.32-22.47 µm), with Mighei showing the largest spectral shift. While the different behavior of Mighei and Murchison might be ascribed to the different energy used in these experiments (40 and 20 keV respectively), more studies will be necessary to elucidate the dependence of the shift on the different meteorite types and/or the different ion energies.

The redshift of the main MIR and FIR bands clearly emerges as a general trend, observed in the bulk spectra of both terrestrial and extraterrestrial silicates. In addition, not only the peaks, but also the spectral minima are shifted, as for instance the minimum observed at about 450 cm -1 (22.2 µm) in CO/CV that shifts towards longer wavelengths after irradiation, as well as the main Christiansen features.

Meteorite

Position before irradiation (cm -1 ) The positions are calculated from the average spectra before and after irradiation with 6x10 16 He + /cm 2 (20 keV for Murchison, 40 keV for the other meteorites).

Cross section of the spectral shift upon irradiation

In the top panel Fig. 3 we report the FIR spectra of three ion irradiation experiments on Allende. The three virgin pellets have similar spectra in terms of shape and band position, which confirms that we are averaging the small scale heterogeneity of the meteorite. We clearly detect the bandshift on all features after irradiation. We note that, no matter the dose, the relative band intensity is modified by irradiation, with bands below 500 cm -1 (above 20 µm) getting stronger compared to the one at 524 cm -1 (19.08 µm).

We studied the relation of the bandshift with the ion fluence on the 524 cm -1 (19.08 µm) band (bottom panel of Fig. 3). We fitted the variation of the peak position (PP) with an equation PP = AP + BS*exp(-σ*F), where AP is the asymptotic position at high irradiation, BS is the asymptotic band shift value, F is the ion fluence and σ is the cross section. We found values AP = 516±1 cm -1 , BS = 7.6±1 cm -1 , and σ=(6±2)x10 -17 cm 2 . We obtained values of correlation coefficient r 2 = 0.99 and reduced 2 = 0.34.

Discussion

Detectability of the irradiation-induced spectral shifts by remote sensing observations

Based on the laboratory evidence of the redshift of IR silicate bands upon irradiation, we explored the possibility of detecting irradiation effects through the remote sensing observations of asteroids, using the bandshift as a proxy. In the top panel of Fig. 4 we display the observed MIR and FIR peak positions for a number of carbonaceous chondrites, before and after irradiation. The peak positions are determined both by the composition of the main minerals and by irradiation. Fortunately, different IR bands provide different proxies of the irradiation process, because they probe different surface layers. Combining the peak positions of at least two bands as in Fig. 4 helps to disentangle the compositional variations from the irradiation effects. A spectral resolution of at least 10 cm -1 seems to be necessary to clearly detect the various spectral shifts produced by ion irradiation.

The separation between the spectral trend linked to the original composition of the meteorites and the irradiation trend is even clearer if the whole IR spectrum is considered. In the bottom panel of Fig. 4, we show the results of the Principal Component Analysis (PCA) of the whole spectral dataset (in the range 100-1200 cm -1 , 8.33-100 µm). A simple 2components PCA is able to separate the experimental points into four main sectors, and thus to easily distinguish anhydrous from hydrated chondrites, and irradiated from unirradiated samples. Similar results are obtained by other clustering methods such as hierarchical clustering or multidimensional scaling (not shown here). The good detection of the irradiation trend by the clustering methods is probably due to the fact that not only the peak positions are affected by irradiation but also the general shape and width of all the IR bands. This demonstrates that measuring a large IR spectral range is a key point for detecting the irradiation effects.

The spectral modifications produced by irradiation proceed rapidly. The irradiation-induced spectral shift increases with increasing ion fluence (see Fig. 3), approaching asymptotic values for fluences above 6x10 16 He + /cm 2 at 40 keV, which corresponds to a timescale of the order of ~10 4 years in the inner Solar System [START_REF] Brunetto | Ion Irradiation of Allende Meteorite Probed by Visible, IR, and Raman Spectroscopies[END_REF]. The use of the laboratory fluences to estimate an astrophysical timescale is justified by the fact that in our experiments we used an ion flux of ~10 13 ions/(sec cm 2 ) that is low enough to statistically avoid an overlapping of the ion tracks (typically less than 10 nm in diameter) during the relaxation of the thermal spikes (typically less than 10 ps in time). A potential dependence of the spectral shift and the corresponding timescale on the ion energy cannot be excluded, and we consider this as an interesting subject for future studies.

Experiments and microscopic analyses suggest that the damage induced by He implantation may be responsible for the spectral modifications of the silicate bands observed in our irradiation experiments [START_REF] Leroux | Spectral and Microstructural Modifications of Olivine Under Ion Irradiation[END_REF]. The implantation of solar wind ions has been identified in Itokawa grains as a major source of stress in the lattice of the original crystalline structure [START_REF] Keller | A Transmission Electron Microscope Study of Itokawa Regolith Grains[END_REF][START_REF] Thompson | Microchemical and Structural Evidence for Space Weathering in Soils from Asteroid Itokawa[END_REF]. In particular, the implanted He creates bubbles and vesicles at the end of the ion tracks, and significant damage all along the irradiated layers [START_REF] Noguchi | Space Weathered Rims Found on the Surfaces of the Itokawa Dust Particles[END_REF]. Previous ion irradiation experiments [START_REF] Brucato | Forsterite Amorphisation by Ion Irradiation: Monitoring by Infrared Spectroscopy[END_REF] showed that ion irradiation of olivine produces an amorphization at high dose, and the authors provided a dependence of the amorphization stage with the ion fluence.

Comparing our fluence values with those reported in the litterature [START_REF] Brucato | Forsterite Amorphisation by Ion Irradiation: Monitoring by Infrared Spectroscopy[END_REF], we estimate that the maximum He fluence we used in our experiments would correspond to a degree of damage in the irradiated layers (about 300 nm penetration depth, [START_REF] Brunetto | Ion Irradiation of Allende Meteorite Probed by Visible, IR, and Raman Spectroscopies[END_REF]) that is about 75% of complete amorphization (see Appendix A for comparison between the penetration depths of ions and photons). We infer that a similar ion-induced surface damage process is taking place on Itokawa and on our meteorites. The efficiency of space weathering in modifying IR optical properties suggests the possibility of detecting this process via IR remote sensing even on relatively young asteroid surfaces. 3). To focus on the effect of different compositions, we include here only meteorites irradiated with exactly the same ion fluence. A Murchison pellet sample, irradiated at lower fluence, provided qualitative similar results. It will be worth comparing the effects of irradiation on different surface preparations in a further dedicated study. Bottom: PC1 and PC2 scores from a principal component analysis performed on the combined MIR and FIR spectra (and baseline corrected, as presented in Fig. 2) of Bennu and the studied CCs. The PC1 and PC2 are not linked to a specific mineralogy, but they rather represent two convenient axes containing the majority of the spectral information. They allow to quickly visualize the spread of our dataset, while reducing its dimensionality. The meteorites data have been resampled to Bennu's resolution for better comparison. Full and open circles are for the measurements before and after irradiation respectively. The color code is the same as the top panel. The arrows give the direction of irradiation. The lines are drawn to highlight the four sectors: unaltered and irradiated hydrated or anhydrous CCs.

Mid-IR and Far-IR spectra have previously been observed on asteroids by telescopes Spitzer [START_REF] Emery | Thermal Emission Spectroscopy (5.2-38 μm) of Three Trojan Asteroids with the Spitzer Space Telescope: Detection of Fine-Grained Silicates[END_REF]) and IRTF [START_REF] Vernazza | Mid-Infrared Spectral Variability for Compositionally Similar Asteroids: Implications for Asteroid Particle Size Distributions[END_REF]. MIR and FIR spectra are currently being measured on asteroid (101955) Bennu by the OTES instrument [START_REF] Christensen | The OSIRIS-REx Thermal Emission Spectrometer (OTES) Instrument[END_REF][START_REF] Hamilton | Evidence for Widespread Hydrated Minerals on Asteroid (101955) Bennu[END_REF]) onboard the OSIRIS-REx sample return mission (NASA) and will soon be observed by the MIRI instrument [START_REF] Rieke | The Mid-Infrared Instrument for the James Webb Space Telescope, I: Introduction[END_REF] of the James Webb Space Telescope (JWST, NASA/ESA). The irradiation-induced spectral shifts are not detectable in IRTF observations because of poor signal to noise ratio (SNR) and spectral resolution (sampling worse than 12 cm -1 at 10 µm), while they are in principle detectable both by the IRS instrument onboard Spitzer and the OTES instrument onboard OSIRIS-REx.

In the case of IRS the spectral resolution was R = λ/∆λ ~ 64-128 and the sampling was about 6.25 cm -1 at 10 µm, while in the case of OTES the spectral sampling was 8.66 cm -1 across the entire spectrum.

The cases of Bennu and Hektor

The top panel of Fig. 4 includes the peak positions observed for the near-Earth asteroid (101955) Bennu [START_REF] Hamilton | Evidence for Widespread Hydrated Minerals on Asteroid (101955) Bennu[END_REF]) and the Trojan asteroid (624) Hektor [START_REF] Emery | Thermal Emission Spectroscopy (5.2-38 μm) of Three Trojan Asteroids with the Spitzer Space Telescope: Detection of Fine-Grained Silicates[END_REF]. Thanks to its proximity to the asteroid, the SNR of the Bennu observation by OTES was about five times better than the SNR of the Hektor observation by IRS (especially in the FIR), which explains the smaller error bars for Bennu in Fig. 4. For comparison, the SNR in our laboratory measurements was about 2000-5000 depending on the spectral range, i.e., at least five times better than OTES. Bennu is reported in the PCA analysis of the bottom panel of Fig. 4, while the lower SNR of Hektor's FIR spectrum does not allow a reliable principal component analysis.

The first OTES observations of Bennu, obtained during the spacecraft approach phase [START_REF] Hamilton | Evidence for Widespread Hydrated Minerals on Asteroid (101955) Bennu[END_REF], provide an interesting test for the use of IR spectra of lab-irradiated carbonaceous chondrites to detect space weathering. Previous ground-based observations of Bennu in the 0.4-2.4 spectral range [START_REF] Clark | Asteroid (101955) 1999 RQ36: Spectroscopy from 0.4 to 2.4μm and Meteorite Analogs[END_REF]) suggested CI-and CM-type chondrites as possible spectral matches for the asteroid. In the first observations performed by the OVIRS instrument onboard OSIRIS-REx in the near-IR spectral range, Bennu showed a clear 2.7 µm hydration band similar to the feature detected in aqueously altered CM1 and CM2 chondrites [START_REF] Hamilton | Evidence for Widespread Hydrated Minerals on Asteroid (101955) Bennu[END_REF]. In terms of mid-and far-IR peak positions observed by OTES (top of Fig. 4), the spectrum of Bennu clearly belongs to the group characterized by the presence of hydrated minerals, as found by [START_REF] Hamilton | Evidence for Widespread Hydrated Minerals on Asteroid (101955) Bennu[END_REF]. The IR peak positions of Bennu are particularly close to the irradiated CM chondrites. Looking in more detail, the PCA reinforces the closeness of Bennu with the lab-irradiated CMs, since its point clearly lies in the sector of the irradiated hydrated meteorites. The proximity of Bennu with the irradiated hydrated meteorites is confirmed by a visual inspection of the spectra (Fig. 5). Although none of the studied meteorites provides a perfect match to Bennu's spectrum, the resemblance of Bennu's spectra with those of irradiated CM Mighei, CM Murchison and potentially C2 Tagish Lake (both virgin and irradiated) is remarkable. Noticeable differences between Bennu and the considered meteorites remain in the right shoulder of the 10-µm feature, on the M-O-H bending region around 600-650 cm -1 , and on the absence of the 290 cm -1 feature in Bennu's spectrum (see also the discussion by [START_REF] Hamilton | Evidence for Widespread Hydrated Minerals on Asteroid (101955) Bennu[END_REF]. The irradiation effects seem to reduce some of these discrepancies. Most importantly, the irradiation significantly improves the match with the positions of all the reststrahlen bands (in particular the bands at ~950 cm -1 and ~440 cm -1 ) and the Christiansen features (see for instance the improved match of the ~530 cm -1 feature), as well as the overall shape of the spectrum. A spectral combination of different irradiated meteorites would get even closer to Bennu in the PCA space. However, the main goal here is not to find the best meteorite match for Bennu, but rather to stress the effect of irradiation on the specific features of the hydrated minerals. The notable match of the diagnostic peak positions of Bennu with those of irradiated meteorites suggests that the surface of Bennu may have recorded the effects of space weathering. If different regions with different degrees of space weathering were present on Bennu, their detection will be possible using OTES data in the near future by more detailed OSIRIS-REx observations, and eventually by the in-depth laboratory studies of the returned samples.

Differently from the case of Bennu, the points relative to Hektor in Fig. 4 are close to those of the anhydrous unirradiated meteorites. This agrees with what has been previously found [START_REF] Emery | Thermal Emission Spectroscopy (5.2-38 μm) of Three Trojan Asteroids with the Spitzer Space Telescope: Detection of Fine-Grained Silicates[END_REF], that olivines dominate the crystalline fraction of Hektor's surface minerals. It is interesting to observe that the peak positions for Hektor are blue-shifted with respect to those of the carbonaceous chondrites of our study (in particular the 11-µm band of olivine). This evidence confirms that none of these meteorites provides a good match of Hektor's spectrum and that the olivines on Hektor are probably richer in Mg than those found in the matrix of Allende, Lancé and FM95002, as it can be deduced based on literature data showing the relation between peak positions and Fo numbers [START_REF] Hamilton | Thermal Infrared (vibrational) Spectroscopy of Mg-Fe Olivines: A Review and Applications to Determining the Composition of Planetary Surfaces[END_REF]. Our results suggest that Hektor's surface has probably suffered less solar wind irradiation than Bennu, which would be in agreement with the dynamical properties of the two asteroids. Considering that the solar wind flux decreases as the square of the distance from the Sun, in the past ten million years [START_REF] Walsh | Craters, Boulders and Regolith of (101955) Bennu Indicative of an Old and Dynamic Surface[END_REF]) a Trojan asteroid like Hektor (semimajor axis a=5.26 AU) must have accumulated an irradiation dose at least 20 times smaller than a near-Earth asteroid like Bennu (semimajor axis a=1.13 AU).

The example of Bennu suggests that irradiation effects may be present also on the primitive near-Earth asteroid (162173) Ryugu. The space weathering of Ryugu has been discussed in the context of visible and near-IR observations by Hayabusa2 [START_REF] Sugita | The Geomorphology, Color, and Thermal Properties of Ryugu: Implications for Parent-Body Processes[END_REF][START_REF] Kitazato | The Surface Composition of Asteroid 162173 Ryugu from Hayabusa2 near-Infrared Spectroscopy[END_REF]), but unfortunately the TIR instrument is not able to detect the MIR-FIR spectral shifts that we measured on irradiated samples. A direct and unambiguous detection of space weathering on Ryugu surface materials will have to wait for the sample return and analysis in 2021.

Will it be possible to detect space weathered surfaces using the JWST? The instrument MIRI has good chances to do so, thanks to its excellent spectral resolution capabilities. The medium resolution spectrometer of MIRI can reach R ~ 2500, a value which would be largely enough to detect the spectral bandshifts induced by irradiation. However, the detection of irradiation effects would still need an adequate signal to noise ratio. Values of SNR > 100-200 (or at least similar to the OTES SNR) seem to be absolutely necessary to unambiguously detect irradiation effects in the spectral range of MIRI. This can probably be achieved by rebinning the data to a lower spectral resolution to increase the SNR.

Conclusions

Spectra collected in the laboratory from virgin and irradiated meteorites indicate that a large IR spectral range is essential to recognize irradiation effects in remote sensing data. Due to their high spectral resolution IR instruments, the new generations of space missions and space telescopes open the way to a clear and unambiguous spectral detection of the space weathering effects on asteroid surfaces. Observations by OSIRIS-REx are now being conducted and future observations by JWST are currently planned in the light of the cycle one proposal to be requested in early 2020. We stress the possibility of detecting space weathering effects with JWST observations, because IR investigation of irradiated surfaces may have broader implications than the study of asteroids. High-quality IR spectra of many unprotected surfaces in the Solar System are -or will soon be -available, from Mercury to the Trans-Neptunian region. As IR spectral variations upon irradiation are less ambiguous than in the VIS-NIR, they are a more reliable source of information to assess the irradiation history of surfaces, and to provide constraints of surface age and geological processes independent from dynamical studies.

On the contrary, the mid-IR (MIR, 2.5-17 µm) and far-IR (FIR, 17-100 µm) spectral ranges show strong absorptions (typical values of k=0.1-10 at the center of the absorption bands [START_REF] Roush | Estimated Optical Constants of the Tagish Lake Meteorite[END_REF][START_REF] Zeidler | TEMPERATURE-DEPENDENT INFRARED OPTICAL CONSTANTS OF OLIVINE AND ENSTATITE[END_REF]), that are due to the high values of the band strengths of the fundamental molecular vibrations of minerals. At the centre of the bands, the main MIR and FIR mineral absorptions probe a much shallower surface layer than VIS-NIR photons.

For instance, considering the optical constants of serpentine [START_REF] Mooney | Optical Constants of Chlorite and Serpentine between 2.5 and 50 μm[END_REF], the penetration depth of visible photons would be higher than 40 µm, whereas it would be about 2 µm for photons in the 2.7 µm OH-band, and it would drop to less than 1 µm for photons from the center of the main MIR and FIR reststrahlen bands. Of course these values change according to the optical properties of the considered target material, but in any case the emerging photons in the main IR bands tend to be the most sensitive to the irradiated layers and are good potential proxies to detect weathered surfaces. The 9-50 µm range, in particular, seems promising because it includes a number of strong absorption bands due to silicates, each one of them providing an independent proxy of the surface. A similar behaviour of wavelength-dependent absorption difference was reported in experimental studies of silica-coated basalts [START_REF] Kraft | Effects of Pure Silica Coatings on Thermal Emission Spectra of Basaltic Rocks: Considerations for Martian Surface Mineralogy[END_REF], which showed that silica coatings can be spectrally detected first in the areas of high absorption, while low absorption areas are only spectrally modified with thicker coatings. An example of detection of irradiation effects using different spectral parameters is reported in Fig. A1. According this this interpretation, we would expect reduced spectral changes from irradiation in the volume scattering areas of low absorption, most particularly in the Christiansen feature areas and transparency regions between the restrahlen bands, and from Figs. 1-3 we see that this is clearly not the case. However, the imaginary index is still quite high in mid-and far-IR regions of low absorption, e.g. k=0.3-1 for serpentine around the Christiansen and transparency features [START_REF] Mooney | Optical Constants of Chlorite and Serpentine between 2.5 and 50 μm[END_REF], and in any case it is higher than the values of k in the visible and near-IR ranges. Other factors that probably need to be considered are how the optical constants are changing within the irradiated layer and how physical changes (vesicularity, lattice stress, etc.) may be affecting the penetration depth. To resolve these observations, future studies will need to combine results from (micro-)spectroscopy and high-resolution transmission electron microscopy of the irradiated layers [START_REF] Leroux | Spectral and Microstructural Modifications of Olivine Under Ion Irradiation[END_REF].

  Fig.1. FIR spectra of six meteorite samples before (black curves) and after (grey) irradiation. Frontier Mountain 95002, Lancé, Tagish Lake, Alais, and Mighei were irradiated with 6x10 16 He + /cm 2 at 40 keV. Murchison spectra were acquired on a polished chip sample irradiated with 6x10 16 He + /cm 2 at 20 keV. Vertical lines are drawn to guide the eye and stress the irradiation effect. The peak positions are reported in Table1and used in Fig.4. All the spectra have been baseline corrected. The intensities are scaled to unity at the position of the main band before irradiation.

Fig. 2 .

 2 Fig. 2. Combined MIR and FIR spectra of ion irradiated carbonaceous chondrites.The continuum-removed reflectance spectra of the meteorites CO3 Frontier Mountain 95002 (top) and C2 Tagish Lake (bottom) are shown before (black curves) and after (grey) laboratory irradiation using 6x10 16 He + /cm 2 at 40 keV. The CO3 and C2 meteorites are chosen here as examples of anhydrous[START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]) and hydrous[START_REF] Nakamura | Mineralogy and Noble-Gas Signatures of the Carbonate-Rich Lithology of the Tagish Lake Carbonaceous Chondrite: Evidence for an Accretionary Breccia[END_REF]) chondrites, respectively. The 830-1100 cm -1 (9-12 µm) and the 200-500 cm -1 (20-50 µm) ranges are characterized by the presence of Si-O stretching and bending vibration bands respectively. In the case of FM 95002, the bands are mainly due to anhydrous minerals (in particular olivine[START_REF] Hamilton | Thermal Infrared (vibrational) Spectroscopy of Mg-Fe Olivines: A Review and Applications to Determining the Composition of Planetary Surfaces[END_REF]). In the case of Tagish Lake, most bands can be attributed to clay minerals (in particular saponite[START_REF] Beck | Transmission Infrared Spectra (2-25μm) of Carbonaceous Chondrites (CI, CM, CV-CK, CR, C2 Ungrouped): Mineralogy, Water, and Asteroidal Processes[END_REF]). In this surface scattering regime, the peaks point towards the top. Vertical lines have been drawn to guide the eye along the position of the main reststrahlen bands and to stress the irradiation effect. The MIR range (1200-650 cm -1 ) has been measured in a previous study[START_REF] Lantz | Ion Irradiation of Carbonaceous Chondrites: A New View of Space Weathering on Primitive Asteroids[END_REF]).

Fig. 3 .

 3 Fig. 3. Irradiation-induced bandshift is an efficient process. Top: Three Allende pellets measured in FIR before (black curves) and after (grey) irradiation at different doses (1, 3, and 6x10 16 He + /cm 2 at 40 keV). The spectra are normalized and shifted for clarity. The peak position before irradiation are highlighted by vertical lines. Bottom: Spectral shift of the 524 cm -1 band in Allende as a function of the ion fluence, along with an exponential fit.

Fig. 4 .

 4 Fig. 4. Comparing spectral parameters of laboratory irradiated meteorites and asteroid observations. Top: MIR and FIR peak positions of carbonaceous chondrites compared to asteroids 101955 Bennu (observed by OTES instrument onboard OSIRIS-REx/NASA (Hamilton et al. 2019)) and 624 Hektor (observed by the IRS instrument on Spitzer (Emery et al. 2006)). The colored arrows describe the space weathering effect (the direction goes from unaltered to irradiated) for each meteorite (Allende, Lancé, Frontier Mountain 95002, Mighei, Alais, Tagish Lake, and Murchison). As shown in Figs. 1-3, Allende, Lancé, and FM95002 have two bands in the 380-540 cm -1 region; so does Hektor. The Murchison data are from the chip. The spectral changes are a function of the ion fluence (see Fig.3). To focus on the effect of different compositions, we include here only meteorites irradiated with exactly the same ion fluence. A Murchison pellet sample, irradiated at lower fluence, provided qualitative similar results. It will be worth comparing the effects of irradiation on different surface preparations in a further dedicated study. Bottom: PC1 and PC2 scores from a principal component analysis performed on the combined MIR and FIR spectra (and baseline corrected, as presented in Fig.2) of Bennu and the studied CCs. The PC1 and PC2 are not

Fig. 5 .

 5 Fig. 5. The spectrum of Bennu compared with unirradiated and irradiated CCs.A linear combination of irradiated CM Mighei and irradiated C2 Tagish Lake meteorites (middle panel) provides a better qualitative match of the average spectrum of Bennu (bottom[START_REF] Hamilton | Evidence for Widespread Hydrated Minerals on Asteroid (101955) Bennu[END_REF])) than the combination of the unirradiated samples (top). The average of the two meteorites is chosen here simply as an example to illustrate the effect of irradiation on the specific features of their hydrated minerals, rather than attempting to find the best analog of Bennu. The peak positions of the main features of Bennu are highlighted by vertical dashed lines. Each spectrum has been normalized to its total intensity.

Fig. A1 .

 A1 Fig. A1. Spectral imaging of irradiated meteorite pellets.A visible image and seven IR spectral parameters maps are reported for laboratory-irradiated carbonaceous chondrites (CV Allende and CM Murchison in this example, top and bottom respectively). The samples were pressed pellets (7 tons, diameter 13 mm) of meteorite powder (unconstrained grain size), and four irradiation areas (diameter 4 mm) were produced at the surface of each sample, using different ions and fluences (Allende: 5E14 and 1E16 He + /cm 2 , and 1E14 and 2E15 Ar + /cm 2 . Murchison: 2E15 and 3E16 He + /cm 2 , and 5E14 and 6E15 Ar + /cm 2 ). Ion irradiation produces an IR spectral shift of the main silicates reststrahlen bands (bottom right map for each meteorite). The IR bandshift induced by irradiation can be used as an irradiation-meter: two irradiation areas (highest fluences for He and Ar) can be easily detected on each meteorite by tracing the peak position of the main silicate band (900 cm -1 for Allende, 1000 cm -1 for Murchison). These irradiation circles are hardly detected by other spectral parameters that use photons in the visible range (upper left visible images) or photons in weaker IR bands. The two other irradiation circles produced at lower fluences are more difficult to recognize, suggesting these fluences are at the detection limit of irradiation effects.

Table 1 . Peak positions and shifts of the main far-IR bands of the studied meteorites.

 1

	Position after	Shift upon irradiation
	irradiation (cm -1 )	(cm -1 )

Acknowledgements

The meteorite samples were kindly provided by the Natural History Museum of Vienna, the Vatican Observatory, J. Brucato, D. Cruikshank, and L. Folco. We are grateful to F. Capitani, A. Celeste, and C. Sandt for their precious help with the bolometer measurements at SOLEIL, and to P. Boiardi for useful suggestions on the manuscript. We warmly thank R.D. Hanna and M.S. Thompson for constructive reviews and discussions. PCA analysis was performed in Quasar (https://quasar.codes) using the Orange Spectroscopy toolbox (Toplak et al. 2017). The irradiations were performed using the INGMAR setup, a joint IAS-CSNSM (Orsay, France) facility funded by the P2IO LabEx (ANR-10-LABX-0038) in the framework Investissements d'Avenir (ANR-11-IDEX-0003-01). The MIR micro-spectroscopy measurements were supported by grants from Region Ile-de-France (DIM-ACAV) and SOLEIL. We thank J.P. Bibring for supporting the FIR spectroscopic activities at SOLEIL. This work has been funded by the Centre National d'Etudes Spatiales (CNES-France, Hayabusa2 mission and OSIRIS-REx mission) and by the ANR project CLASSY (Grant ANR-17-CE31-0004-02) of the French Agence Nationale de la Recherche.

SUPPLEMENTARY MATERIALS Appendix A: detection of space weathering

The detectability of space weathering by reflectance spectral measurements is a function of the photon wavelength and of the surface optical properties, because it requires that the photons probe mainly the weathered layer of the upper surface. In the case of the slow solar wind, the irradiated layer is thinner than 1 µm [START_REF] Noguchi | Space Weathered Rims Found on the Surfaces of the Itokawa Dust Particles[END_REF][START_REF] Brunetto | Ion Irradiation of Allende Meteorite Probed by Visible, IR, and Raman Spectroscopies[END_REF]. From the spectroscopic studies of minerals and meteorites, it is known that the VIS-NIR spectral region is characterized by the presence of weak absorptions, with typical values of k=10 -4 -10 -2 for the imaginary index [START_REF] Roush | Estimated Optical Constants of the Tagish Lake Meteorite[END_REF][START_REF] Brunetto | Optical Characterization of Laser Ablated Silicates[END_REF]. In these conditions, photons interact with the surface materials in a volume scattering regime [START_REF] Salisbury | Mid-Infrared Spectroscopy: Laboratory Data[END_REF], probing layers from tens to hundreds of µm thick, which are deeper than the penetration depth of solar wind ions. In the reflectance measurements of an irradiated surface, the emerging light would thus bring mixed information of both altered and unaltered layers, in a relative ratio which depends in a non-linear way on the absorption coefficient.


Eman Assrawi

Camille Louvrier

Clémence Lepelletier

Sophie Georgin-Lavialle

Jean-David Bouaziz

Fawaz Awad

Florence Moinet

Philippe Moguelet

Marie Dominique Vignon-Pennamen

William Piterboth

Marie Dominique Vignon-Pennamen

Claire Jumeau

Laetitia Cobret

Elma Elkhouri

Bruno Copin

Philippe Duquesnoy

Marie Legendre

Gilles Grateau

Sonia A Karabina

Serge Amselem
email: serge.amselem@inserm.fr

Irina Giurgea
email: irina.giurgea@inserm.fr

Orcid Eman Assrawi

Serge Amelem

Somatic Mosaic NLRP3 Mutations and Inflammasome Activation in Late-Onset Chronic Urticaria

Keywords: Chronic Infantile Neurological, Cutaneous and Articular syndrome CRP: C-Reactive Protein EV: empty vector FCAS: Familial Cold Autoinflammatory Syndrome HEK293T: Human Embryonic Kidney cell line IL1β: Interleukin 1β IL1R: Interleukin 1 Receptor LRR: Leucine-Rich Neutrophilic Urticarial Dermatosis SNHL: Sensorineural Hearing Loss chronic urticaria, neutrophilic urticaria, NLRP3-assocaited autoinflammatory disease, NLRP3, somatic mosaic mutations, NLRP3-inflammasome activation, anti-interleukin 1 receptor antagonists

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

INTRODUCTION

Urticarial rash is a common dermatological disorder characterized by sudden and recurrent onset of usually pruritic cutaneous wheals with pale central swelling, surrounded by skin erythema and edema, affecting about 15-25% of individuals [START_REF] Kanani | Urticaria and angioedema[END_REF]). Hives appears over any part of the body and individual lesions usually last for a few hours and up to 24 hours [START_REF] Criado | Chronic urticaria in adults: state-ofthe-art in the new millennium[END_REF]. In its acute form, the urticarial rash lasts for less than 6 weeks, while in the chronic form the rash extends over 6 weeks in a continuous or intermittent manner [START_REF] Kaplan | Pathogenesis of chronic urticaria[END_REF]. Chronic urticaria is more common in adults, with a peak age of onset between 20 and 40 years, and can be associated with significant morbidity and decreased quality of life. In the absence of physical triggers, chronic urticarial rash is called idiopathic or spontaneous [START_REF] Zuberbier | The EAACI/GA(2) LEN/EDF/WAO Guideline for the definition, classification, diagnosis, and management of urticaria: the 2013 revision and update[END_REF][START_REF] Beck | A Review of International Recommendations for the Diagnosis and Management of Chronic Urticaria[END_REF]. Allergy, autoimmune and infectious disorders can underlie chronic urticaria, a symptom that requires extensive tests for identifying the causative factors [START_REF] Beck | A Review of International Recommendations for the Diagnosis and Management of Chronic Urticaria[END_REF]. Urticarial rash can also be one of the symptoms of NLRP3-associated autoinflammatory disease (NLRP3-AID), a group of rare monogenic systemic autoinflammatory disorder previously known as Cryopyrin-Associated Periodic Syndrome (CAPS) [START_REF] Kuemmerle-Deschner | Diagnostic criteria for cryopyrin-associated periodic syndrome (CAPS)[END_REF][START_REF] Ben-Chetrit | Consensus proposal for taxonomy and definition of the autoinflammatory diseases (AIDs): a Delphi study[END_REF]. NLRP3-AID is clinically a heterogeneous syndrome and consists of three overlapping entities with increasing severity: (i) the familial cold autoinflammatory syndrome (FCAS, OMIM #120100) characterized by neonatal onset of cold-induced inflammatory episodes of fever, arthralgia and urticarial rash; (ii) the Muckle-Wells syndrome (MWS, OMIM #191900) characterized by infantile-to-childhood onset of recurrent attacks of fever, urticarial skin rash and arthritis with subsequent sensorineural hearing loss (SNHL); and (iii) the chronic infantile neurological, cutaneous and articular syndrome (CINCA, OMIM #607115) characterized by infantile onset of chronic persistent clinical disease course of ongoing fever, central nervous system involvement, continuous urticarial skin rash and deformative joint arthritis [START_REF] Jb | CAPS--pathogenesis, presentation and treatment of an autoinflammatory disease[END_REF].

NLRP3, the disease-causing gene is expressed mainly in myeloid blood cells, but also in B and T lymphocytes (Hoffman et al. 2001a;[START_REF] Sutterwala | Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1[END_REF]) and in keratinocytes which could therefore account for the predisposition of patients for urticarial rash [START_REF] Kummer | Inflammasome components NALP 1 and 3 show distinct but separate expression profiles in human tissues suggesting a site-specific role in the inflammatory response[END_REF][START_REF] Dai | Epidermal keratinocytes sense dsRNA via the NLRP3 inflammasome, mediating interleukin (IL)-1β and IL-18 release[END_REF]. The encoded NLRP3 protein (also known as cryopyrin) comprises an N-terminal pyrin domain (PYD), a central nucleotide-binding and oligomerization domain (NACHT, standing for NAIP, CIITA, HET-E and TP1), a NACHT-associated domain (NAD) and a C-terminal leucine rich repeats (LRR). NLRP3 initiates the assembly of a multiprotein complex called inflammasome [START_REF] Martinon | The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta[END_REF][START_REF] Schroder | The inflammasomes[END_REF] through the following steps: NLRP3 interacts with the adaptor protein ASC (Apoptosis-associated speck-like protein containing a CARD) leading to the assembly of ASC dimers into multimers designated as ASC specks [START_REF] Fernandes-Alnemri | The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation[END_REF]. ASC specks, the hallmark of inflammasome activation, form a scaffold for recruitment and subsequent activation of procaspase-1 into caspase-1. Active caspase-1 cleaves proinflammatory cytokines, mainly IL1β, into their mature and secreted forms, which orchestrates the systemic inflammatory response [START_REF] Martinon | The inflammasomes: guardians of the body[END_REF].

NLRP3 mutations identified in NLRP3-AID patients result in a gain-of-function effect leading to NLRP3-inflammasome activation. Initially described in affected families as autosomal dominant disorders due to heterozygous NLRP3 mutations (Hoffman et al. 2001a), NLRP3-AID is now also known as a sporadic disease due to de novo germline or somatic mutations. Primarily identified in leukocytes from pediatric patients [START_REF] Saito | Somatic mosaicism of CIAS1 in a patient with chronic infantile neurologic, cutaneous, articular syndrome[END_REF][START_REF] Saito | Diseaseassociated CIAS1 mutations induce monocyte death, revealing low-level mosaicism in mutation-negative cryopyrin-associated periodic syndrome patients[END_REF][START_REF] Aróstegui | A somatic NLRP3 mutation as a cause of a sporadic case of chronic infantile neurologic, cutaneous, articular syndrome/neonatal-onset multisystem inflammatory disease: Novel evidence of the role of low-level mosaicism as the pathophysiologic mechanism underlying mendelian inherited diseases[END_REF][START_REF] Tanaka | High incidence of NLRP3 somatic mosaicism in patients with chronic infantile neurologic, cutaneous, articular syndrome: results of an International Multicenter Collaborative Study[END_REF][START_REF] Omoyinmi | Whole-Exome Sequencing Revealing Somatic NLRP3 Mosaicism in a Patient With Chronic Infantile Neurologic, Cutaneous, Articular Syndrome[END_REF][START_REF] Nakagawa | Somatic NLRP3 mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes[END_REF][START_REF] Lasigliè | Cryopyrinassociated Periodic Syndromes in Italian Patients: Evaluation of the Rate of Somatic NLRP3 Mosaicism and Phenotypic Characterization[END_REF], somatic mosaic NLRP3 mutations have been more recently found in some adult patients with a typical NLRP3-AID phenotype [START_REF] Zhou | Brief Report: Cryopyrin-Associated Periodic Syndrome Caused by a Myeloid-Restricted Somatic NLRP3 Mutation[END_REF][START_REF] Mensa-Vilaro | Brief Report: Late-Onset Cryopyrin-Associated Periodic Syndrome Due to Myeloid-Restricted Somatic NLRP3 Mosaicism[END_REF][START_REF] Rowczenio | Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism-UK Single Center Experience[END_REF].

We describe here two unrelated elderly patients presenting for two decades with a chronic urticarial skin rash resistant to standard therapy and associated with systemic inflammation during the rashes. Two different mosaic NLRP3 mutations were identified by means of targeted sequencing using next generation (NGS) technologies. To identify the origin of these mutational events, we studied the mosaicism level in several cell types from both patients. In addition, to assess the pathogenicity of the identified variations, we studied their functional consequences using different in vitro assays.

RESULTS

Disease phenotype (Table 1)

Proband I, a 65-year-old man, had recurrent episodes of diffuse non-pruritic fleeting urticarial skin rash involving the trunk and limbs (Figure 1a). The rash was intermittent, lasted for a few hours (4-5 hours) and repeated every 2-3 days since the age of 49 years.

No triggers like cold exposure, physical exercise, stress, nutrition or medications were noted. The urticarial lesions were accompanied with high-grade fever (39C°), chills, and flu-like symptoms including arthralgia, fatigue and headache. There was no vasculitis, adenopathy, organomegaly or ophthalmological, abdominal or neurological manifestations.

During the crises, C-reactive protein (CRP) was elevated (20 mg/L). Immunological and infectious profile, serum protein electrophoresis and kidney function tests were normal.

Skin biopsy revealed urticarial lesions with a rich inflammatory infiltrate composed mostly of neutrophils with no signs of vasculitis (Figure 1c).

Proband II, a 67-year-old woman, developed the first episode of chronic urticarial skin rash at the age of 46 years. Urticarial lesions manifested as migratory transient erythematous plaques (Figure 1b), with no pruritus. Distributed over the whole body (thighs, abdomen, thorax, upper limbs and face), the rash, which were not triggered by cold, lasted fewer than 12 hours and occurred two to three times per week. It was associated with high-grade fever (39-40C°), chills, arthritis, myalgia, headache, adenopathy and hepatomegaly. No other abdominal or ophthalmological manifestations were observed. During the attacks, CRP levels ranged from 70 to 120 mg/L. At the age of 50 years, she developed bilateral SNHL. A skin biopsy revealed the presence of dermal neutrophilic infiltration with no alteration of capillaries and no signs of vasculitis (Figure 1d). Kidney function tests were normal and extensive analyses of the immunological and infectious profile were found negative. Serum protein electrophoresis revealed polyclonal hypergammaglobulinemia.

None of the patients had family history of skin, autoimmune or autoinflammatory disorders. In both probands, second generation H1-antihistamines (the standard therapy), oral corticosteroids and colchicine showed no efficacy.

Molecular analyses

With the aim to identify the etiology of the chronic recurrent urticaria that occurred in a systemic inflammatory context, we collected the genomic DNA from whole blood samples of Probands I and II. We looked for systemic autoinflammatory disorders (SAIDs) through targeted sequencing of the main genes so far involved in these conditions. In both patients, somewhat unexpectedly, somatic mosaic mutations in NLRP3 exon 3 were identified.

Proband I carried an in-frame deletion (c.926_934del p.(Gly309_Phe311del)), and Proband II a missense variation (c.1705G>A p.(Glu569Lys)). The mosaicism level in the whole blood DNA for c.926_934del was of 17.2% and of 11% for the c.1705G>A transition (Figure S1).

The in-frame deletion of three amino acids (Gly309, Ala310 and Phe311) identified in Proband I is located within the NACHT domain of NLRP3 (Figure 2a) in close proximity to the Walker B motif of the protein, which is a Mg2 + -binding loop implicated in ATP hydrolysis and essential for NLRP3-inflammasome activation [START_REF] Neven | Molecular basis of the spectral expression of CIAS1 mutations associated with phagocytic cell-mediated autoinflammatory disorders CINCA/NOMID, MWS, and FCU[END_REF]).

The glutamic acid residue at position 569 (Glu569), involved in the mutation identified in Proband II (p.Glu569Lys), belongs to the NAD domain of the NLRP3 protein, a domain of so far unknown function (Figure 2a). The identified mutations involved evolutionarily conserved amino acids in mammals (Figure 2b). Moreover, both the NLRP3 transcript, including exon 3 that contains these NLRP3 mutations (https://ecrbrowser.dcode.org) and the encoded regions are uniformly conserved across mammalian species [START_REF] Duéñez-Guzmán | The evolution of reproduction-related NLRP genes[END_REF]Lv et al. 2017).

Modelization of the NLRP3 structure, based on a Nod2 crystal [START_REF] Maekawa | Crystal structure of NOD2 and its implications in human disease[END_REF], showed that both the three deleted amino acids and Glu569 are located close to the Walker B motif (Figure 2c).

Cellular distribution of NLRP3 mosaicism

To study the cellular distribution of the NLRP3 mosaicism, different cell types were subsequently isolated from Probands I and II. The analyzed cells include monocytes, neutrophils, B and T cells that have a mesodermal embryonic origin, urinary epithelial cells that are representative of the endodermal embryonic cells and buccal cells that are of ectodermal origin. Targeted sequencing showed a wide distribution of the mutated alleles in the studied cells (Figure 3, lower panel and Figure S1). We took advantage of these data to look for the two identified NLRP3 variants by Sanger sequencing. This study revealed the presence of a superimposed sequence, mimicking a background noise, but corresponding to those mutations (Figure 3, upper Panel). Mutated alleles were detected in monocytes, neutrophils and B cells at average levels of 13% in Proband I and 12% in Proband II. The T cells showed a proportion of the mutated allele of 10% for Proband I and of 5% for Proband II. In buccal cells, the proportion of the mutated allele was of 3.8% for Proband I and it was estimated around 5% (using Sanger sequencing due to the low amount of DNA available) for Proband II. In the urinary epithelial cells, the proportion of the mutated allele was estimated around 5% (using Sanger sequencing) in both probands.

Impact of NLRP3 mutations on ASC speck formation

To assess the pathogenicity of the identified NLRP3 mutations, we tested their effects on NLRP3 function by studying ASC speck formation, a common readout of inflammasome activation (Man and Kanneganti, 2015). To this end, we transfected HEK293T (ASC-GFP_C1-FLAG) cells with plasmids encoding wild-type (WT) NLRP3 or NLRP3 carrying the deletion (pNLRP3-Del) or the missense variation (pNLRP3-Glu569Lys), or with an empty vector (EV). In agreement with the known role of NLRP3 in inflammasome assembly and ASC speck formation, cells transfected with pNLRP3-WT displayed a significantly higher percentage of ASC specks as compared to cells transfected with the EV alone. Importantly, cells transfected with the pNLRP3-Del or pNLRP3-Glu569Lys construct showed a significantly higher percentage of ASC specks as compared to cells transfected with pNLRP3-WT, thereby revealing a gain-of-function effect of the two identified mutations on inflammasome activation (Figure 4 and Figure S2).

Impact of NLRP3 mutations on IL1β secretion

To test the impact of the p.(Gly309_Phe311del) and p.(Glu569Lys) mutations on cytokine secretion, we used THP1 cells, which express endogenously NLRP3, ASC, procaspase-1 and IL1β. THP1 cells were first primed with PMA (Phorbol 12-Myristate 13-Acetate) in order to obtain an adherent macrophage-like phenotype and to induce IL1B gene expression. Cells were then transfected with pNLRP3-WT, pNLRP3-Del, pNLRP3-Glu569Lys or the EV and treated with lipopolysaccharide (LPS), a well-known activator of the NLRP3 inflammasome. IL1β secretion, a hallmark of inflammasome activation, was assessed in the cell culture supernatants. IL1β levels were found to be significantly higher in the supernatant of cells transfected with pNLRP3-Del or pNLRP3-Glu569Lys as compared to cells transfected either with pNLRP3-WT or with the EV (Figure 5).

Treatment

Consistent with the above-described data showing NLRP3-inflammasome activation, both probands showed complete remission with the anti-IL1R antagonist Anakinra (100mg/day) administered by subcutaneous injections. One week after Anakinra administration, both patients reported a significant clinical and biological improvement. A complete resolution of the urticarial skin rash, the fever and the systemic inflammation (CRP levels <5mg/L) was observed. Probands I and II are still under treatment today, having received the treatment for the past two and three years, respectively. During the follow-up period, kidney function tests were found to be normal in both probands. However, the treatment, has so far no effect on hearing loss.

DISCUSSION

We described two unrelated patients presenting long-term chronic urticarial skin rash of late-adulthood onset. The clinical presentation of the patients is reminiscent of neutrophilic urticarial dermatosis (NUD), a condition characterized by an urticarial rash with predominate neutrophilic infiltration at skin biopsy and systemic inflammation [START_REF] Kieffer | Neutrophilic urticarial dermatosis: a variant of neutrophilic urticaria strongly associated with systemic disease. Report of 9 new cases and review of the literature[END_REF][START_REF] Gusdorf | Neutrophilic urticarial dermatosis: A review[END_REF]. For two decades, our tow patients were refractory to anti-histamines, steroids and colchicine and, despite broad investigations, the underlying etiology was not identified. In both patients, the results of serum protein electrophoresis strongly argued against the diagnosis of Schnitzler syndrome, which is mainly characterized by the simultaneous occurrence of monoclonal gammopathy and chronic urticaria [START_REF] Gusdorf | Schnitzler Syndrome: a Review[END_REF]. Taken together, these data prompted us to look for possible gene mutations through the use of a targeted sequencing approach using NGS technology focusing on the main genes so far implicated in autoinflammatory disorders. Somewhat unexpectedly, this analysis identified in both patients a somatic mosaic NLRP3 variation, raising the question of a NLRP3-AID diagnosis in the two patients. The diagnosis of NLRP3-AID is indeed challenging for several clinical and molecular reasons.

Firstly, mostly reported in children (either in familial forms or sporadic cases), the clinical diagnosis of NLRP3-AID is puzzling in the absence of a contributive family history and in adult patients with late-onset chronic urticaria occurring in the context of systemic inflammation. Although the occurrence of deafness in such context should evoke the diagnosis of NLRP3-AID, hearing loss is common with aging. Of note, SNHL, which is a hallmark of MWS, was present in both probands. It was identified at the age of 50 years for Proband II, while, for Proband I, it was identified at the age of 65 years by means of audiometric tests that were performed once the NLPR3 mutation was found.

Secondly, regarding the molecular investigations, it is important to underline that mosaic mutations are usually undetectable by Sanger sequencing. Targeted sequencing using NGS technologies increases the sensitivity of detection of such somatic mutations and high-depth sequencing is necessary to detect low level mosaicisms. To further determine the cellular distribution of the mutated alleles, we analyzed the DNA obtained from patients' cells derived from different embryonic germ layers. A wide cell-type distribution was observed for both mutations suggesting that the mutational events arose early during human embryogenesis. Similarly, a wide tissue distribution of the mutated mosaic NLRP3 alleles has been also reported in five patients with a late-onset typical NLRP3-AID [START_REF] Rowczenio | Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism-UK Single Center Experience[END_REF]. In patients carrying mosaic NLRP3 mutations, gonadal cells were not studied for obvious reasons. Consequently, vertical transmission to the offspring cannot be excluded. The mechanism underlying the late-disease onset in patients carrying widely distributed somatic mutations is so far not understood. Variable age of disease onset was also observed in patients with germline NLRP3 mutations [START_REF] Hoffman | Familial cold autoinflammatory syndrome: phenotype and genotype of an autosomal dominant periodic fever[END_REF]. As suggested by different authors [START_REF] Yu | Cryopyrin-associated periodic syndrome: an update on diagnosis and treatment response[END_REF][START_REF] Giat | Cryopyrin-associated periodic syndrome[END_REF][START_REF] Walker | Brief Report: Severe Inflammation Following Vaccination Against Streptococcus pneumoniae in Patients With Cryopyrin-Associated Periodic Syndromes[END_REF], external factors such as cold, stress or exercise could trigger the disease development.

Finally, to demonstrate the pathogenicity of newly identified NLRP3 variants and, therefore, to establish the diagnosis of NLRP3-AID, functional studies are mandatory. Indeed, NLRP3 variants do not always affect the protein function. The mosaic variations identified in our patients are located in two regions considered as hot spots for NLRP3 mosaicism [START_REF] Nakagawa | Somatic NLRP3 mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes[END_REF]. The in-frame deletion (p.Gly309_Phe311del) is located in the region of the NACHT domain (305-309 amino acids) that contains 8 out of the 47 reported mosaic mutations. The p.(Glu569Lys) variant is located in the NAD domain (565-572 amino acids) that includes half of the reported mosaic mutations (n=22/47). The threedimensional (3D) model of NLRP3 showed that these two hot spots including the in-frame deletion and the p.(Glu569Lys) variation are close to the Walker B motif, thereby suggesting their pathogenicity. Using in vitro assays, we found significantly higher ASC speck formation and IL1β secretion in cells expressing the NLRP3 mutants identified in our patients, thereby clearly demonstrating the pathogenicity of these mutations.

In conclusion, our data support the idea that in chronic urticaria, the presence of systemic inflammatory markers should evoke a NLRP3-related disorder. Beyond the search for NLRP3 mutations, functional assays demonstrating a gain-of-function effect of those variants are essential to confirm their pathogenicity. Consistent with mutations triggering NLRP3-inflammasome activation, complete remission can be achieved with antiinterleukin 1 receptor antagonists, thereby, highlighting the importance to identify the underlying cause of chronic urticaria in those patients.

PATIENTS AND METHODS

Patients

Clinical features were recorded on a standardized form. Informed written consent for genetic studies was given by the participants. Skin punch biopsy was performed on recent urticarial lesions after local anesthesia. Skin sections were stained with haematoxylin, eosin and saffron (HES) and examined under a light microscope.

Molecular analyses

Genomic DNA was extracted from peripheral blood leukocytes of Probands I and II using standard procedures. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Pancoll density gradient. PBMCs were separated by positive selection using immunomagnetic microbeads (MACS, Miltenyi Biotec), anti-CD14 (monocytes) followed by anti-CD15 (neutrophils), anti-CD19 (B cells) and anti-CD3 (T cells). DNA was then extracted from monocytes, neutrophils, B cells, T cells, urine and buccal cells using proteinase K digestion followed by phenol-chloroform extraction protocol. The DNA samples were sequenced following a targeted sequencing approach using a custom targeted capture (SeqCap EZ system, Roche) on a NextSeq500 or MiSeq (Illumina) platform as previously described [START_REF] Jeanson | Mutations in GAS8, a Gene Encoding a Nexin-Dynein Regulatory Complex Subunit, Cause Primary Ciliary Dyskinesia with Axonemal Disorganization[END_REF]. The position of the target oligos is available on request. We used a custom targeted capture which contains the coding and the untranslated regions (UTR) of the following genes: ADA2, CARD14, IL1RN, IL36RN, LACC2, LPIN2, MEFV, MVK, NLRC4, NLRP12, NLRP3, NOD2, PLCG2, PSMB8, PSTPIP1, RBCK1, TMEM173, TNFAIP3, TNFRSF11A and TNFRSF1A.

Sequence data were analyzed using an in-house double pipeline with sequence reads in fastq format aligned to the reference human genome (hg19) using BWA and Bowtie2.

Duplicate sequence reads were removed (by Picard and SAMtools, respectively). Variant calling was performed using GATK and SAMtools respectively. Additional screening of indels was performed by the Pindel software. Variant calls in VCF format were then annotated through ANNOVAR. All results obtained by this targeted sequencing were confirmed using Sanger sequencing. NLRP3 mutation numbering was based on the cDNA sequence (NM_004895).

Modelization of the NLRP3 structure

The 3D structure of the human NLRP3 protein has been modeled as follows: (i) using the HHpred software, we found that the 3D structure of the NACHT, NAD and LRR domains of Nod2 (5IRN, Oryctolagus cuniculus) [START_REF] Maekawa | Crystal structure of NOD2 and its implications in human disease[END_REF]) has been identified as the closest to human NLRP3 protein; (ii) using the Modeller software, the predicted structure of human NLRP3 has been obtained and (iii) using the Pymol software, the amino acids involved in mosaic NLRP3 mutations have been highlighted.

Plasmid constructs

Human NLRP3 cDNA was subclonned into the expression plasmid (pcDNA3.1/V5-His-TOPO, Invitrogen) to generate the wild-type expression vector (pNLRP3-WT) as described previously [START_REF] Jéru | PYPAF1 nonsense mutation in a patient with an unusual autoinflammatory syndrome: role of PYPAF1 in inflammation[END_REF]). Site-directed mutagenesis was subsequently performed to generate plasmids carrying either the deletion identified in Proband I (p.Gly309_Phe311del) designated as (pNLRP3-Del) or the previously reported p.(Glu569Lys) mutation that we identified in Proband II (pNLRP3-Glu569Lys), using the "quick change site-directed mutagenesis kit" (Stratagene, Le Jolla, CA). The resulting plasmid constructs were confirmed by Sanger sequencing.

Cell culture and transfection

HEK293T cells stably expressing FLAG-tagged procaspase-1 (procaspase-1-FLAG) and green fluorescent protein (GFP)-tagged-ASC (designated ASC-GFP and C1-FLAG) kindly provided by Professor Emad Alnemri (Thomas Jefferson University, Philadelphia, PA), were cultured in DMEM-F12 (Gibco). HEK293T cells (ASC-GFP and C1-Flag) were transfected with 375 ng or 500 ng of the following plasmids: pNLRP3-WT, pNLRP3-Del, pNLRP3-Glu569Lys, or with the EV using FuGENE-HD (Promega) for 24h. THP1 cells (ATCC ® -TIB202) were initially primed with 100 ng/ml PMA (Phorbol 12-Myristate 13-Acetate) for 3h, then transfected with 500 ng of pNLRP3-WT, pNLRP3-Del, pNLRP3-Glu569Lys, or with the EV using the FF-100 program in the 4D-Nucleofector™, Amaxa™ (Lonza) for THP1 cells. Following transfection, cells were treated with LPS 100 ng/ml and incubated at 37C°for 24h. At the indicated time, supernatants of transfected cells were collected and stored at -80°C for IL1β measurement by ELISA.

Speck quantification assay

Following HEK293T cells (ASC-GFP_C1-FLAG) transfection, the formation of ASC aggregates (specks) was calculated 24 hours later. Manual counting of cells containing ASC-GFP specks was performed in 5 representative randomly selected fields at 20× magnification. The percentage of specks was calculated as the number of specks divided by the total number of counted cells for three independent experiments. Cells were observed using a Nikon Eclipse TS100 inverted fluorescent microscope.

Statistical analyses

Differences were analyzed statistically using the unpaired student t-test and were plotted with the GraphPad Prism software. A p value <0.05 was considered as statistically significant.

Figure 1 .

 1 Figure 1. Urticarial lesions and skin biopsy

Figure 2 .

 2 Figure 2. NLRP3 mutations and their impact at the protein level

Figure 3 .

 3 Figure 3. NLRP3 DNA sequencing in different cell types

Figure 4 .

 4 Figure 4. Impact of NLRP3 mutations on ASC speck formation

Figure 5 .

 5 Figure 5. Impact of NLRP3 mutations on IL1β secretion

Acknowledgments

We are grateful to the affected persons and their families whose cooperation made this study possible. We would like to thank Pastelle Metura and the M2 Bioinformatics group of Paris University for their help in developing a 3D NLRP3 model. This study was funded by the Agence Nationale de la Recherche ANR-17-CE17-0021-01. FA was supported by Alquds University in Palestine and from the "Fondation pour la Recherche Médicale" (FDT20130928419). EA was supported by An-Najah University in Palestine. CLo was supported by INSERM (Poste d'accueil, 00552404).

Data availability statement

All the data sets related to this study are included in the article.

Conflict of interest

GG declares fees less than 5000 euros from Novartis, BMS and SOBI. The remaining authors have no conflict of interest to declare.

Author contributions

Conceptualization: IG and SA. Data curation: EA and CLo. Formal analysis: EA and CLo.