Hamed Vahdati Nasab

Gilles Berillon
email: gilles.berillon@mnhn.fr

Guillaume Jamet

Milad Hashemi

Mozhgan Jayez

Somaye Khaksar

Zohreh Anvari

Guillaume Guérin

Maryam Heydari

Mohammad Akhavan Kharazian

Simon Puaud

Stéphanie Bonilauri

Valéry Zeitoun

Noémie Sévêque

Darvishi Khatooni

Asgari Asghar

Khaneghah

Marcel Otte

Désert Central Iranien

Mirak

The open-air Paleolithic site of Mirak, northern edge of the Iranian Central Desert (Semnan, Iran): Evidence of repeated human occupations during the late Pleistocene

Keywords:

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

Introduction

The wide area extending from the Zagros-Caucasus to Central Asia is historically acknowledged as a key region for understanding prehistoric human settlements, from the first human dispersal into Eurasia during the early Pleistocene to the upper Pleistocene [START_REF] Zeitoun | Where palaeoanthropology and prehistory meet[END_REF], including the transition from Middle Paleolithic to Upper Paleolithic cultures (e.g., [START_REF] Becerra-Valdivia | Chronometric investigations of the Middle to Upper Paleolithic transition in the Zagros Mountains using AMS radiocarbon dating and Bayesian age modelling[END_REF][START_REF] Coon | Cave Explorations in Iran1949[END_REF][START_REF] Heydari-Guran | The MUP Zagros Project: tracking the Middle Upper Paleolithic transition in the Kermanshah region, West-central Zagros, Iran[END_REF][START_REF] Mcburney | Preliminary Report on Stone Age Reconnaissance in north-eastern Iran[END_REF][START_REF] Otte | Iran Paleolithic/Le Paléolithique d'Iran. UISPP[END_REF][START_REF] Otte | New radiocarbon dates for the Zagros Aurignacian from Yafteh cave[END_REF][START_REF] Smith | Paleolithic Archaeology in Iran[END_REF]. However, despite the discoveries of numerous Paleolithic sites in this region and broad interest in the transition from Upper to Middle Paleolithic, to date, few archaeological assemblages have been obtained using accurate geological and chronological frameworks. These are mainly concentrated on the mountainous areas, especially the Zagros (Fig. 1) and dated to the MIS3 (see [START_REF] Becerra-Valdivia | Chronometric investigations of the Middle to Upper Paleolithic transition in the Zagros Mountains using AMS radiocarbon dating and Bayesian age modelling[END_REF] for the current chronological framework). Some of them include: Shanidar [START_REF] Solecki | Shanidar cave: a Paleolithic site in northern Iraq[END_REF], Warwasi [START_REF] Braidwood | Mousterian large-mammal remains from Kobeh Cave behavioral implications for Neanderthals and early modern humans[END_REF][START_REF] Tsanova | The beginning of the Upper Paleolithic in the Iranian Zagros. A taphonomic approach and techno-economic comparison of Early Baradostian assemblages from Warwasi and Yafteh (Iran)[END_REF], Yafteh [START_REF] Hole | The Prehistory of South-western Iran: a preliminary report[END_REF][START_REF] Otte | The Aurignacian of Yafteh Cave and its context (2005-2008 excavations)[END_REF], Gar Arjeneh [START_REF] Hole | The Prehistory of South-western Iran: a preliminary report[END_REF][START_REF] Otte | Témoins aurignaciens dans le Zagros, Iran[END_REF], Ghar-e Khar [START_REF] Shidrang | Continuity and change in the late Pleistocene lithic industries of the central Zagros: a typotechnological analysis of lithic assemblage from Ghar-e Khar cave, Bisotun. Iran[END_REF][START_REF] Young | Research in the prehistory of central western Iran[END_REF], Guilvaran and Kaldar [START_REF] Bazgir | Test excavations and initial results at the Middle and Upper Paleolithic sites of Gilvaran, Kaldar, Ghamari caves and Gar Arjene rockshelter, Khorramabad Valley, western Iran[END_REF][START_REF] Bazgir | Understanding the emergence of modern humans and the disappearance of Neanderthals: Insights from Kaldar Cave (Khorramabad Valley, western Iran)[END_REF][START_REF] Roustaei | New Research the Paleolithic of Lurestan, West Central Iran[END_REF], Gh ār-e Boof [START_REF] Ghasidian | Excavations at Ghar-e Boof in the Fars Province of Iran and its Bearing on Models for the Evolution of the Upper Paleolithic in the Zagros Mountains[END_REF][START_REF] Heydari | Paleolithic survey in southwestern Zagros, Basht Valley[END_REF]. Other sites have been discovered in the Zagros and along its eastern fringes during the last decade. Most of these sites lack an adequate chronological framework, such as Mar-Tarik and Qaleh Bozi [START_REF] Biglari | Recent finds of Paleolithic period from Bisotun, Central Western Zagros Mountains[END_REF][START_REF] Biglari | Test excavations at the Middle Paleolithic sites of Qaleh Bozi, Southwest of Central Iran[END_REF][START_REF] Jaubert | Paléolithique moyen récent de la grotte de Qaleh Bozi 2 (Ispahan, Iran) : premiers résultats de la campagne 2008[END_REF][START_REF] Jaubert | The Middle Paleolithic occupation of Mar-Tarik, a new Zagros Mousterian site in Bisotun massif (Kermanshah, Iran)[END_REF] as well as Zavyeh, Qaleh Gusheh and Holabad (Heydari-Guran et al., 2015). Several open-air sites have been discovered in the central Alborz and the northern edge of the Iranian Central Desert: the Upper Paleolithic site of Garm Roud [START_REF] Berillon | Discovery of new open-air Paleolithic localities in Central Alborz, northern Iran[END_REF](Berillon et al., , 2016) ) dated to the end of the MIS3, and the surface sites of Mirak [START_REF] Rezvani | Settlement patterns of prehistoric cultures in Semnan[END_REF][START_REF] Vahdati Nasab | Late Pleistocene dispersal corridors across the Iranian Plateau: A case study from Mirak, a Middle Paleolithic site on the northern edge of the Iranian Central Desert (Dasht-e Kavir)[END_REF], Moghanak and Otchunak [START_REF] Berillon | Discovery of new open-air Paleolithic localities in Central Alborz, northern Iran[END_REF][START_REF] Chevrier | Moghanak, Otchounak, Garm Roud 2 : nouveaux assemblages paléolithiques dans le Nord de l'Iran. Caractérisations typo-technologiques et attributions chrono-culturelles[END_REF]Chevrier et al., , 2010)), Delazian [START_REF] Vahdati Nasab | The Upper Paleolithic of the Iranian Central Desert: the Delazian Sites, Semnan Province-a Case Study[END_REF], and Chah-e Jam [START_REF] Vahdati Nasab | Playas and Middle Paleolithic settlement of the Iranian Central Desert: The discovery of the Chah-e Jam Middle Paleolithic site[END_REF]. The location and cultural affinities of these sites suggest that the northern fringes of the Iranian Central Desert could have constituted a corridor used during upper Pleistocene hunter-gatherer dispersals [START_REF] Vahdati Nasab | Late Pleistocene dispersal corridors across the Iranian Plateau: A case study from Mirak, a Middle Paleolithic site on the northern edge of the Iranian Central Desert (Dasht-e Kavir)[END_REF].

In that chronocultural context, our interest focused on the open-air site of Mirak, which is located 16 km to the south of the outskirts of the city of Semnan (Fig. 2). First mentioned by [START_REF] Rezvani | Settlement patterns of prehistoric cultures in Semnan[END_REF], Mirak was officially surveyed in 2009 by one of the authors (HVN) as part of the Paleolithic Survey of the Iranian Central Desert Project (PSICDP) [START_REF] Rezvani | A major Middle Paleolithic open site at Mirak, Semnan Province, Iran[END_REF]. This survey collected a large lithic assemblage from the surface of eight natural mounds with evident Middle Paleolithic affinities (flake-based blank production, an abundance of prepared and chapeau de gendarme platforms, a significantly high value for the Levallois index, the presence of tools typical of 'Mousterian' technology). Only scarce artifacts with Upper Paleolithic affinities were found at the site, which suggests either the presence of Upper Paleolithic techno-complex and/or the presence of transitional lithic industries at the site [START_REF] Vahdati Nasab | Late Pleistocene dispersal corridors across the Iranian Plateau: A case study from Mirak, a Middle Paleolithic site on the northern edge of the Iranian Central Desert (Dasht-e Kavir)[END_REF]. Mound 8, though not the largest mound in Mirak, exhibits the greatest volume of lithic scatters on its surface and surroundings. Moreover, the presence of relatively dense archaeological material visible in spoil heaps around one of the clandestine looters' pits suggests that some archaeological deposits of Middle and Upper Paleolithic affinities were still in situ, buried, and protected by the mound.

Based on the foregoing, there was a need for a systematic excavation in this vicinity. Based upon our preliminary observations, Mirak appeared to be potentially very informative. Therefore, three seasons of excavation at Mirak 8 were conducted under the framework of joint Iranian-French collaboration (May-June 2015, July-August 2016, and October-November 2017). The following presents the preliminary results of these excavations, concerning the nature of archaeological deposits at Mirak, their cultural affinities, and their geological and absolute chronological framework. Finally, these original data provide new perspectives with regards to the human settlements of the region during the MIS3.

Results and discussion

The excavation and finds

The excavations at Mirak 8 exposed an area covering 36 m 2 in total, divided between three main sectors (Fig. 3): 19 m 2 on the northern slope of the mound, 12 m 2 on the eastern slope and 5 m 2 on the southern one, delimited based on geoarchaeological prospecting. The excavation was carried out by squares with a surface area of 1 m 2 each, using both contextual and arbitrary level excavation techniques. The conventional depth of 5 cm was adopted for each arbitrary archaeological level, taking into account the depositional settings, and each excavated archaeological find was collected and referenced in the 3D frame of the mound. Dry and wet sievings of sediments were used when possible in order to sample micro-artifacts. The excavation exposed an excavated surface totaling 19 m 2 (north sector, 4 m 2 ; east sector, 10 m 2 ; south sector, 5 m 2 ).

The three campaigns of excavation yielded 6266 finds, including 2709 objects, precisely collected in the stratigraphic context and spread between 4 and 7 m below the top of the stratigraphic sequence. These archaeological materials are mainly composed of lithic artifacts (see below), but rare and extremely altered fragments of bones and teeth of large mammals were also recovered; no sign of human intervention on these altered materials could be identified. The determinable teeth fragments belong to equids. No remains of small fauna were found either in excavation contexts or in the sieved sediments.

We focus here on the results of the excavation on the eastern trench, where the archaeological density appeared to be the highest. Three main successive archaeological concentrations were clearly identified in stratigraphic context (Layers 1-3) (Fig. 5).

Depositional setting and chrono-stratigraphic framework of Mirak 8 2.2.1. The Mirak stratigraphic sequence

Mirak is located in a dry mudflat (53 • 25'53" E; 35 • 28'10" N; ca. 1035 masl) that extends from Semnan's piedmont along the southern flank of the Alborz Mountains to the northern edge of the Iranian Central Desert, also known as Dasht-e Kavir. This area belongs to an elongated Quaternary drainage system, which is characterized by alluvial fans and sedimentary pediments, with widespread saline playa-lake systems downstream. Geomorphological fieldwork has revealed a pedosedimentary succession represented by a 9-m-thick alluvial-aeolian record made up of two main sequences (Sequence I: alluvial basal complex; Sequence II: windblown deposits) (see Fig. 4 for the synthetic stratigraphy and description). Sequence I can be divided into six lithostratigraphic units from the base upwards (Units 9-4; thickness: ca. 5 m). Horizontal bedding of silty clays (Units 8, 6 and 4) were observed in the stratigraphy. This stratigraphic column also includes at least two bodies of very fine sands (Units 7 and 5) and a deflated horizon (Unit 4a); these three units contain the three main archaeological assemblages (3 to 1 respectively). Sequence I accumulated on a pediplain that was flooded repeatedly as a consequence of shallow sheet flooding and channel overflows. Indeed, horizontal bedding prevails in the very fine sand units (Units 7 and 5), but current ripple marks and internal planar cross bedding occur as well (lithofacies Sr and Sp according to Miall 1996). The whole units are distinguished by intense post-depositional weathering, resulting both from the repetition of decreases in the level of the water table and increases in the rate of evaporation, as evidenced by Fe-Mn concretions, iron hydroxide precipitates, calcareous nodules, gypsum crystals, and desiccation cracks. These features are associated with the development of 'Aridisol horizons' (Bk, By) (USDA, Soil Survey Staff, 2014). The upper limit of this alluvial pedocomplex is marked by a major discontinuity, which corresponds to the contemporary deflation surface of Dasht-e Kavir. Sequence II of Mirak 8 mound can be divided into four lithostratigraphic units from the base upwards (Units 3-0; thickness: about 4 m). The first part of this sequence consists of calcareous aeolian sand deposits (Unit 3) reworked by a hydromorphic horizon (Unit 2) topward. The uppermost part of Sequence II is composed of another aeolian sand deposit (Units 1 and 0) affected by the development of an entisol (USDA).

Luminescence dating

Samples for luminescence dating were collected: six samples were taken from the northern section of Sequences I and II and two samples from the lower part of the eastern section of Sequence I. Preliminary dating results, including analytical data, are listed in Table 1 and plotted in their stratigraphic position in Fig. 4 (see also Supplementary Material for details on the implemented procedure). OSL and post-IR IRSL measurements were performed on multigrain aliquots (see SM for methodological details). Overall, the OSL ages are coherent with the stratigraphy and the inter-aliquot dispersion in equivalent doses for each sample is very low; this allows us to consider the OSL ages to provide accurate, reliable ages. As a result, the ages range from 50 ± 3 to 28 ± 2 ka in the Sequence I that contains the three primary archaeological deposits, with a maximum age of 28 ± 2 ka for Layer 1, dates between 28 ± 2 and 38 ± 2 Light brown sandy silt with massive structure (entisol developed on calcareous windblown deposits). 2. Convoluted light-grey sandy silt including lenses of reworked wind-blown deposits (unit 1) (hydromorphic horizon). 3. Brown clayey silt with massive structure (calcareous windblown deposits). 4a. Pale green silty clay with polyhedral structure (non-calcareous floodplain fines-hydromorphic horizon). 4b and 6. Greyish green silty clay with prismatic structure and Fe-Mn oxide coatings (non-calcareous floodplain fines). 5 and 7. Pale green silty sand with current ripples (Sr) and internal planar crossbedding (Sp) (shallow water deposits and minor channel fills). 8. Dark greyish green silty clay with prismatic structure including Fe-Mn oxide coatings, very fine sands and small pebbles of grey sandstone (non-calcareous floodplain fines and crevasse splay deposits). 9. Dark brown silty clay (Sketch by G. Jamet). This synthetic stratigraphy was built up on the basis of the correlation between three vertical sections (North, East & South trenches) and 14 sediment logs from Auger surveys and clandestine holes. Fig. 4. Stratigraphie synthétique de Mirak 8 (2016) et localisation des âges OSL/IRSL. 0. Sol arable à structure plate (surface végétalisée de la bute). 1. Silt sableux brun clair à structure massive (entisol développé sur des dépôts calcaires soufflés par le vent). 2. Silt sableux gris clair convoluté comprenant des lentilles de dépôts remaniés par le vent (unité 1) (horizon hydromorphique). 3. Silt argileux brun à structure massive (dépôts calcaires éoliens). 4a. Argile silteuse vert pâle à structure polyédrique. 4b et 6. Argile silteuse vert grisâtre à structure prismatique et revêtements d'oxyde de Fe-Mn. 5 et 7. Sable silteux vert pâle avec des ondulations de courant (Sr) et lits entrecroisés planaires internes (Sp) (dépôts d'eau peu profonde et remplissages mineurs de chenaux). 8. Argile silteuse vert grisâtre foncé, à structure prismatique, comprenant des revêtements d'oxyde de Fe-Mn, du sable très fin et de petits cailloux de grès gris (dépôts fins et craquelés non calcaires de plaine d'inondation). 9. Argile limoneuse brun foncé (dessin de G. Jamet). Cette stratigraphie synthétique a été construite sur la base de la corrélation entre trois sections verticales (tranchées nord, est et sud) et 14 diagraphies sédimentaires provenant de levés à la tarière et de sondages clandestins.

for Layer 2, dates between 47 ± 2 ka and 47 ± 4 ka for Layer 3 (samples MK 16/2 and MK16/3), which correspond to the Last Glacial cycle. The ages range from 1.2 ± 0.2 to 0.6 ± 0.1 ka in Sequence II.

Formation of the mound of Mirak 8 and context of archaeological deposits

Based on the available data, it seems that Sequence I at Mirak Mound 8 may have formed as a response to climatic changes during the first half of the Last Glacial (probably during MIS 3), which was more humid than the Upper Pleniglacial (MIS 2 age equivalent). The records of these transitional periods are less well understood in the context of the northern Iranian paleoclimatic and chronostratigraphic framework for the Last Interglacial/Glacial cycle (i.e. the Lake Urmia core: [START_REF] Djamali | A late Pleistocene long pollen record from Lake Urmia, Iran[END_REF]; loess records of North of Iran: [START_REF] Kehl | Loess paleosol-sequences along a climatic gradient in northern Iran[END_REF][START_REF] Frechen | Loess chronology of the Caspian Lowland in northern Iran[END_REF]. Human occupations at Mirak were contemporaneous with the aggradation of a covered pediment incised by anastomosed channels. This period was apparently characterized by rapid flooding events (i.e. sheet flood or crevasse splay deposits) that buried Paleolithic deposits. If the increase in rainfall events explains the accumulation of sediments deposited during flash floods, we do not exclude the influence of episodic uplift upstream, as pointed out by [START_REF] Kehl | Quaternary climate change in Iran-the state of knowledge[END_REF]. Nevertheless, no stratigraphic evidence of tectonic effect has been observed. Aggradation of the alluvial cover ended with a major aridification marked by an aridisol development, perhaps indicating the end of the Last Glacial period. The scenario of a long-term drying coincided with a sedimentary hiatus, between the units 4 and 3, which separated the Pleistocene desiccated flat from the Holocene sand dune (sequence II). The Holocene history of Mirak is defined by at least two phases of calcareous dust deposits interrupted by the temporary development of a perched water body on the first aeolian mound. The formation of this hydromorphic horizon (Unit 2) in arid environment implies a groundwater flux from the bedrock and its resurgence by a perched spring.

The study of the excavated sediments evidently indicates that, in contrast to the current arid desert situation, the northern part of the Iranian Central Desert, at least for the studied region, had a completely different climate during the end of the Pleistocene. During the first half of the Last Glacial Period (MIS 4/3) the climate of the region was cold and humid. This cold and humid period was characterized by a slow alluvial aggradation interrupted by rapid flooding events. Two of the archaeological layers at Mirak 8 are dated to the chronological interval between 50 ± 3 ka and 28 ± 2 ka, which corresponds to these humid climate conditions. Moreover, the different nature of the sediments of the archaeological layers in comparison to the upper and lower sterile sediments implies that, during each of these occupational events (which might have lasted hundreds of years), the site was situated on dry land, possibly surrounded by streams and marshes. Concerning the Holocene sequence (Sequence 2 in Mirak 8), the current study of Mirak 8 demonstrates that the mounds in Mirak cannot be seen as remnants of a more extensive deposit that has been eroded away, but correspond to Holocene nebkha or shadow dunes. In Mirak 8, it results in at least two phases of calcareous dust deposits interrupted by the temporary development of a perched water body on the first aeolian mound. The two OSL ages derived from the very top of the mound (MK15/1: 0.6 ± 0.1ka) and where the mound meets the flatland area around it (MK15/4: 1.2 ± 0.2 ka) indicate that the formation of the mound of Mirak 8 took approximately 800-1600 years. Therefore, the mounds of the site were formed at a relative rapid pace. As mentioned above, such hydromorphic horizon in arid environment implies a groundwater flux from the bedrock and its resurgence by a perched spring. In that general pattern of dune formation, the vegetation may have played an important role in the formation of the mound by fixing the calcareous sands and consequently the preservation of the underlying Pleistocene sequence, as originally proposed by Vahdati Nasab et al. ( 2013).

Lithics and cultural affinities

The lithic assemblage

The lithic assemblage presented here consists of 2313 pieces from the easternmost excavation at Mirak 8. More than 85% of these artifacts are made of varieties of chert (i.e. chert, flint, and jasper). Other raw materials are tuff, sandstone, siltstone, and rarely, microconglomerate. Within this assemblage, 976 pieces are typologically indeterminable fragments or splinters, most of which have very small dimensions; their techno-typological identification is problematic, if not impossible (see [START_REF] Jayez | An experimental study on the methods of quantifying fragmented flake[END_REF] for the morphological characteristics of what is considered "indeterminable"). These pieces have been excluded from our analysis, which leaves 1337 chipped stones to present, including 212 chips (Table 2), which are flakes mostly with dimensions of less than 1 cm (see Shea, 2013: 32).

The three identified levels occurred in distinct stratigraphic units, although some vertical dispersion are observed within each level that may be related to some taphonomic agents (work in progress) (Fig. 5); however, the general state of preservation of the lithics is very good, which testifies to the fact that, if taphonomy has impacted the deposits, the impact has been limited. In that respect, Level 1 appears to occur in a deflated unit; this may explain the wider dispersion and lower density in objects than those observed for levels 2 and 3. In addition, few artefacts were located between these three main archaeological deposits, usually in whole roots or vertical brakes. Apart these very few pieces (around 30 among 2313 artefacts), the artefacts appear to depict three main archaeological deposits.

The L1 lithic assemblage appears to be an industry oriented toward a blade/bladelet production from at least carinated elements. No cores have been found and other tool types are underrepresented. Carinated burins are found exclusively in L1 (Fig. 6: 1-3), which are usually found in the so-called "Zagros Aurignacian" (see: [START_REF] Olszewski | The Zarzian Occupation at Warwasi Rockshelter, Iran[END_REF]Dibble, 1994, 2002) or "Baradostian" (see: [START_REF] Olszewski | The Zarzian Occupation at Warwasi Rockshelter, Iran[END_REF][START_REF] Solecki | The Baradostian Industry and the Upper Paleolithic in the Near East[END_REF] techno-complexes. The characteristic artifact-types of this assemblage include blades, bladelets, and their associated fragments (Fig. 6:4), although Arjeneh (Font-Yves) points and lamelles Dufour as major components of both central Zagros and southern Zagros Upper Paleolithic lithic industries (see [START_REF] Conard | The Rostamian cultural group and the taxonomy of the Iranian Upper Paleolithic[END_REF][START_REF] Ghasidian | Excavations at Ghar-e Boof in the Fars Province of Iran and its Bearing on Models for the Evolution of the Upper Paleolithic in the Zagros Mountains[END_REF][START_REF] Hole | The Paleolithic Culture Sequence in western Iran[END_REF][START_REF] Hole | The Prehistory of South-western Iran: a preliminary report[END_REF][START_REF] Otte | The Aurignacian in the Zagros Region: New Research at Yafteh Cave, Lorestan. Iran[END_REF] are not observed in the current assemblage of Mirak. Among the platform rejuvenation flakes in this assemblage is one large core tablet (Fig. 6: 5) that indicates the large dimensions of prismatic cores from which the blades were produced. Finally, Levallois-Mousterian tools are completely absent in this layer. As a whole, this layer shows Upper Paleolithic affinities. However, this assessment must be taken with caution; indeed, the density of artifacts is low (see Table 2) and the assemblage was preserved in a deflated horizon and thus may not be fully representative of the original one. The L2 lithic assemblage is characterized by the mixed presence of Upper Paleolithic industries with characteristics of prismatic blade/bladelet production and Levallois-Mousterian flake-based production, the technological structure of which shows also a moderate percentage of cores and tools (Table 2). Few blades and bladelets are either plain or regularly retouched or notched (Table 4, Fig. 7: 4-5). Semi-parallel arrises (flake scar ridges) are present on the dorsal face of some blades (Fig. 7: 3). The Mousterian industry artifact types are dominated by the Levallois technique (for Mousterian artifact-types see, e.g., [START_REF] Geneste | Analyse lithique d'industries moustériennes du Périgord : approche technologique du comportement des groupes humains au Paléolithique moyen[END_REF]. They mostly indicate recurrent reduction strategy with radial/centripetal preparation (Fig. 7:6). Levallois flakes with facetted and chapeau de gendarme platforms are present as well as the byproducts of Levallois reduction strategy in the form of core preparation elements. Bladelets and some of the blades have been made via the prismatic reduction method. It is worth mentioning that Levallois blade cores are rare in the Mirak assemblage. Regarding the tools (Table 4), among the interesting specimens is one elongated convergent blade with facetted striking platform and inverse notching/retouching (Fig. 7: 1). As a whole, Layer 2 appears to present a mix of characteristics usually associated with both Upper and Middle Paleolithic industries.

In L3, cores and tools are well represented (twice as much as in upper layers). Most of the cores are Levallois cores (Fig. 8: 11). If the debitage composition of the layers is considered, L3 lithic assemblage, with less than 5% blade and bladelet products, indicates an industry based on flake production, a number of them being relatively elongated (Table 3). This assemblage includes Mousterian industry artifact types similar to L2 with the dominance of Levallois technique with mostly recurrent reduction strategy with radial/centripetal preparation. Regarding the tools (Table 4), Levallois-Mousterian tools such as various scrapers (Fig. 8: 8, 10) are present in high quantity in L3 as well as some points (Fig. 8: 2, 5-7); they are comparable to those found in the Zagros Mousterian (see [START_REF] Dibble | Le Paléolithique moyen récent du Zagros[END_REF][START_REF] Dibble | The Middle Paleolithic Industries of Warwasi[END_REF][START_REF] Lindly | The Zagros Mousterian: A regional perspective[END_REF]. Some tools have basal trimming, which might be attributed to the hafting methods (Fig. 8: 1,8). There are also Levallois points with retouch on both faces and facetted striking platform (Fig. 7: 9). As a whole, L3 has clear Middle Paleolithic affinities.

However, the vertical distribution and field data (such as post-depositional reworking) shows that some pieces cannot be precisely allocated to Layers 2 or 3; among these pieces, one carinated scraper as well as a prismatic core (Fig. 8: 3), here vertical between L2 and L3 respectively, are usually considered as a typical implement of Upper Paleolithic industry of Baradostian/Zagros Aurignacian (see [START_REF] Olszewski | To be or not to be Aurignacian: The Zagros Upper Paleolithic[END_REF][START_REF] Otte | The Aurignacian in the Zagros Region: New Research at Yafteh Cave, Lorestan. Iran[END_REF].

Phases of human occupation

In contrast to what has been claimed previously about the Middle Paleolithic affinities based on the study of the surface collection of the lithic industries in Mirak [START_REF] Rezvani | A major Middle Paleolithic open site at Mirak, Semnan Province, Iran[END_REF][START_REF] Vahdati Nasab | Late Pleistocene dispersal corridors across the Iranian Plateau: A case study from Mirak, a Middle Paleolithic site on the northern edge of the Iranian Central Desert (Dasht-e Kavir)[END_REF], the data derived from the three seasons of excavations at Mirak 8 has clearly identified the presence of vestiges of at least three main archaeological layers.

The upper layer, i.e. L1, has clear Upper Paleolithic affinities, with some resemblance (carinated burins) with the so-called "Zagros Aurignacian" [START_REF] Olszewski | The Zarzian Occupation at Warwasi Rockshelter, Iran[END_REF]Dibble, 1994, 2002) or "Baradostian" [START_REF] Olszewski | The Zarzian Occupation at Warwasi Rockshelter, Iran[END_REF][START_REF] Solecki | The Baradostian Industry and the Upper Paleolithic in the Near East[END_REF] techno-complexes. Yet Arjeneh (Font-Yves) points and lamelles Dufour as major components of both central Zagros and southern Zagros Upper Paleolithic lithic industry (see [START_REF] Conard | The Rostamian cultural group and the taxonomy of the Iranian Upper Paleolithic[END_REF][START_REF] Ghasidian | Excavations at Ghar-e Boof in the Fars Province of Iran and its Bearing on Models for the Evolution of the Upper Paleolithic in the Zagros Mountains[END_REF][START_REF] Hole | The Paleolithic Culture Sequence in western Iran[END_REF][START_REF] Hole | The Prehistory of South-western Iran: a preliminary report[END_REF][START_REF] Otte | The Aurignacian in the Zagros Region: New Research at Yafteh Cave, Lorestan. Iran[END_REF] are not observed in the current assemblage of Mirak; if confirmed, Mirak would more closely resemble Garm Roud, which dates back to around 33 kya [START_REF] Berillon | Discovery of new open-air Paleolithic localities in Central Alborz, northern Iran[END_REF](Berillon et al., , 2016;;[START_REF] Chevrier | Moghanak, Otchounak, Garm Roud 2 : nouveaux assemblages paléolithiques dans le Nord de l'Iran. Caractérisations typo-technologiques et attributions chrono-culturelles[END_REF]. However, the low density of artifacts in L1 and its evident reworked nature does not allow a precise determination of this upper assemblage and needs caution in its interpretation. The Upper Paleolithic affinities are consistent with the OSL dating, which gives a maximum age of 28 ± 2 kya, and with the presence of a large Upper Paleolithic open-air site, i.e. Delazian, at a very close proximity (2 km northeast of Mirak, see Vahdati Nasab and Clark, 2014).

The lower layer, i.e. L3, has clear Middle Paleolithic affinities; with regards to Levallois-Mousterian tools (various scrapers and points); the assemblage is comparable to those of classical Zagros Mousterian sites (see [START_REF] Dibble | Le Paléolithique moyen récent du Zagros[END_REF][START_REF] Dibble | The Middle Paleolithic Industries of Warwasi[END_REF][START_REF] Lindly | The Zagros Mousterian: A regional perspective[END_REF]. Interestingly, this The intermediate layer, i.e. L2, might be the most controversial one, representing a mixture of industries with both Middle and Upper Paleolithic affinities, in a chronological framework that dates it to between 28 ± 2 and 38 ± 2 kya. Based on this preliminary chronological framework, L2 appears possibly contemporary of the early Upper Paleolithic of the Zagros [START_REF] Becerra-Valdivia | Chronometric investigations of the Middle to Upper Paleolithic transition in the Zagros Mountains using AMS radiocarbon dating and Bayesian age modelling[END_REF], where facetted platforms or sidescrapers have been identified (e.g., [START_REF] Tsanova | The beginning of the Upper Paleolithic in the Iranian Zagros. A taphonomic approach and techno-economic comparison of Early Baradostian assemblages from Warwasi and Yafteh (Iran)[END_REF]. However, more studies are ongoing, including the technotypology of the intermediate layer, as well as a refined chronology based on recent OSL samples that should allow us refining our understanding of the nature, the chronology and the context of this intermediate human occupations at Mirak. However, to our knowledge, despite some potential resemblances with the early Upper Paleolithic of the Zagros, such mixture of industries has no equivalent to date within the regional context of the Iranian Central Plateau, and together with its chronological framework points the complexity of the cultural evolution in a wide area between Zagros and Central Asia.

Finally, beside this general pattern, a carinated scraper and a prismatic core, which are typical of the Baradostian/Zagros Aurignacian (see [START_REF] Olszewski | To be or not to be Aurignacian: The Zagros Upper Paleolithic[END_REF][START_REF] Otte | The Aurignacian in the Zagros Region: New Research at Yafteh Cave, Lorestan. Iran[END_REF], have been collected between the lowest layers (L2 and L3) and could not be allocated precisely to one of these levels. Although, as mentioned above, this may be related to some post-depositional reworking based on field data and vertical distribution, this points to the originality of the lithic industries of Mirak, and one cannot exclude some potential affinities of the Mirak's lower lithic assemblages with early Upper Paleolithic or late Middle Paleolithic cultures of the Zagros. Further analysis of the lithic assemblage will address these potential affinities.

Conclusion

In summary, Mirak provides unique data in the area between the Zagros and Central Asia, depicting successive

Fig. 1 .

 1 Fig. 1. Map of some of the major Paleolithic sites in the studied area.

Fig. 1 .

 1 Fig. 1. Carte des sites paléolithiques majeurs de la région d'étude.

Fig. 2 .

 2 Fig. 2. Northern Iranian Central Desert and Mirak 8.

Fig. 2 .

 2 Fig. 2. Le Nord du Désert central iranien et la butte Mirak 8.

Fig. 3 .

 3 Fig. 3. Topographic map of Mirak 8.

Fig. 3 .

 3 Fig. 3. Carte topographique de Mirak 8.

Fig. 4 .

 4 Fig. 4. Synthetic stratigraphy of Mirak 8 in 2016 and location of the OSL/IRSL dating. 0. Topsoil with platy structure (vegetalized surface of the mound). 1.Light brown sandy silt with massive structure (entisol developed on calcareous windblown deposits). 2. Convoluted light-grey sandy silt including lenses of reworked wind-blown deposits (unit 1) (hydromorphic horizon). 3. Brown clayey silt with massive structure (calcareous windblown deposits). 4a. Pale green silty clay with polyhedral structure (non-calcareous floodplain fines-hydromorphic horizon). 4b and 6. Greyish green silty clay with prismatic structure and Fe-Mn oxide coatings (non-calcareous floodplain fines). 5 and 7. Pale green silty sand with current ripples (Sr) and internal planar crossbedding (Sp) (shallow water deposits and minor channel fills). 8. Dark greyish green silty clay with prismatic structure including Fe-Mn oxide coatings, very fine sands and small pebbles of grey sandstone (non-calcareous floodplain fines and crevasse splay deposits). 9. Dark brown silty clay (Sketch by G. Jamet). This synthetic stratigraphy was built up on the basis of the correlation between three vertical sections (North, East & South trenches) and 14 sediment logs from Auger surveys and clandestine holes. Fig.4. Stratigraphie synthétique de Mirak 8 (2016) et localisation des âges OSL/IRSL. 0. Sol arable à structure plate (surface végétalisée de la bute). 1. Silt sableux brun clair à structure massive (entisol développé sur des dépôts calcaires soufflés par le vent). 2. Silt sableux gris clair convoluté comprenant des lentilles de dépôts remaniés par le vent (unité 1) (horizon hydromorphique). 3. Silt argileux brun à structure massive (dépôts calcaires éoliens). 4a. Argile silteuse vert pâle à structure polyédrique. 4b et 6. Argile silteuse vert grisâtre à structure prismatique et revêtements d'oxyde de Fe-Mn. 5 et 7. Sable silteux vert pâle avec des ondulations de courant (Sr) et lits entrecroisés planaires internes (Sp) (dépôts d'eau peu profonde et remplissages mineurs de chenaux). 8. Argile silteuse vert grisâtre foncé, à structure prismatique, comprenant des revêtements d'oxyde de Fe-Mn, du sable très fin et de petits cailloux de grès gris (dépôts fins et craquelés non calcaires de plaine d'inondation). 9. Argile limoneuse brun foncé (dessin de G. Jamet). Cette stratigraphie synthétique a été construite sur la base de la corrélation entre trois sections verticales (tranchées nord, est et sud) et 14 diagraphies sédimentaires provenant de levés à la tarière et de sondages clandestins.

Fig. 5 .

 5 Fig. 5. Mirak 8. Vertical distribution of the archaeological finds, eastern sector; pecks of high density (black arrows). The projection of finds in the east-west vertical plane highlights the three main archaeological concentrations observed during the excavation. They represent a sub-horizontal slope, with a slight dip upwards near the periphery of the mound. The upper assemblage depth (L1) is around 100 ± 20 cm above the reference level. The intermediate assemblage (L2) is the thickest, with a depth around 20 ± 30 cm below the reference level. The basal assemblage (L3) is around 115 ± 12 cm below the reference level. Fig. 5. Mirak 8. Distribution verticale des objets archéologiques, secteur est ; pics de densité (flèches noires). La projection des objets dans le plan vertical est-ouest met en évidence les trois principales concentrations archéologiques observées lors des fouilles. Subhorizontale, vers l'ouest, elles présentent un léger pendage vers le haut près de la périphérie du monticule. L'assemblage supérieur (L1) est à environ 100 ± 20 cm au-dessus du niveau de référence. L'assemblage intermédiaire (L2) est le plus épais, à une profondeur d'environ 20 ± 30 cm en dessous du niveau de référence, au centre de la butte (lignes O & P). L'assemblage profond (L3) est à environ 115 ± 12 cm sous le niveau de référence.

Fig. 6 .

 6 Fig. 6. Lithic artifacts from L1. 1-3. Carinated burin. Twisted blade. 5. Core tablet (drawings by M. Jayez).

Fig. 6 .

 6 Fig. 6. Industrie lithique de L1. 1-3. Burins carénés. 4. Lame torse. 5. Tablette (dessins de M. Jayez).

  assemblage dates between 47 ± 2 kya and 47 ± 4 kya, which, although compatible with the general chronological framework of Middle Paleolithic assemblages in the Middle East, appears very recent compared to the Middle Paleolithic known in the Zagros, and earlier than the early Upper Paleolithic assemblages of the Zagros (Becerra-Valdivia et al., 2017).

Fig. 7 .

 7 Fig. 7. Lithic artifacts from L2. 1. Mousterian point on a blade. 2. End scraper. 3-4. Bladelet fragments. 5. Notched blade fragment. 6. Recurrent centripetal flake core (drawings by M. Jayez). Fig. 7. Industrie lithique de L2. 1. Pointe moustérienne sur lame. 2. Grattoir terminal. 3-4. Fragments de lamelle. 5. Fragment de lame avec encoche. 6. Nucléus récurrent, centripète à éclats (dessins de M. Jayez).

Fig. 8 .

 8 Fig. 8. Lithic artifacts from L3. 1. Notch-denticulated. 2. Convergent point/scraper? 3. Prismatic core (L2-L3). 4. Levallois blade. 5. Déjeté point. 6-7. Mousterian point on elongated flake. 8. End scraper with basal trimming on the ventral face. 9. Levallois point. 10. Side scraper and notched. 11. Levallois flake core (drawings by M. Jayez). Fig. 8. Industrie lithique de L3. 1. Denticulé. 2. Pointe/grattoir ? convergent 3. Nucléus prismatique (L2-L3). 4. Lame Levallois. 5. Pointe déjetée. 6-7. Pointes moustériennes sur éclat allongé. 8. Grattoir terminal avec retouche basale sur la face ventrale. 9. Pointe Levallois. 10. Grattoir latéral et à encoche. 11. Nucléus Levallois à éclat (dessins de M. Jayez).

Table 1

 1 Summary of quartz OSL and feldspar post-IR IRSL data.

	Tableau 1
	Données OSL du quartz et post-IR IRSL du feldspath.
	Sample	Quartz OSL					Feldspar post-IR IRSL
		Depth a (m)	n	De (Gy)	Dose rate (Gy•ka -1 )	Age (ka)	n	De (Gy)	Dose rate (Gy•ka -1 )	Age (ka)
	MK15/1	0.65	20	1.2 ± 0.2	2.18 ± 0.05	0.6 ± 0.1	15	4.4 ± 0.3	2.51 ± 0.07	1.7 ± 0.1
	MK15/4	3.5	18	2.9 ± 0.4	2.39 ± 0.04	1.2 ± 0.2	15	7.6 ± 0.3	2.72 ± 0.07	2.8 ± 0.1
	MK15/5	4.1	25	92 ± 4	3.29 ± 0.07	28 ± 1	1 5	118 ± 4	3.62 ± 0.09	32 ± 2
	MK15/6	4.55	25	95 ± 6	3.37 ± 0.06	28 ± 2	14	116 ± 3	3.70 ± 0.08	31 ± 1
	MK15/8	5.70 b	25	135 ± 6	3.60 ± 0.05	38 ± 2	14	197 ± 4	3.93 ± 0.07	50 ± 2
	MK15/7	6.2	25	159 ± 6	3.16 ± 0.09	50 ± 3	14	249 ± 9	3.49 ± 0.10	71 ± 4
	MK16/2	-1.10	19	123 ± 4	2.63 ± 0.08	47 ± 2	15	193 ± 8	2.99 ± 0.08	65 ± 4
	MK16/3	-1.18	20	156 ± 11	3.32 ± 0.10	47 ± 4	15	454 ± 52	3.68 ± 0.10	123 ± 17
	Notes. 'De' is the average equivalent dose and 'n' is the number of the measured aliquots.
	a Compared to the top of the stratigraphy.
	b MK15/8, sampled in S2 at 4.60 m below the top of the S2 stratigraphy.

Table 2

 2 Technological structure of the Mirak chipped stone assemblage (L = Layer; Lithic density = total quantity of chipped stones/excavated volume in m 3 ). Structure technologique de l'assemblage de pierres concassées de Mirak (L = couche ; Lithic density = quantité totale de pierres concassées/volume extrait en m 3 ).

	Tableau 2
		L1	%	L2	%	L3	%	Sum
	Core and frags	0	0	12	1.8	27	4.2	39
	Tools	10	18.2	121	18.7	225	35.5	356
	Debris	0	0	13	2	12	1.9	25
	Debitage	45	81.8	502	77.5	370	58.4	917
	Sum	55	100	648	100	634	100	1337
	+Indeterminable Fragments	13		496		467		976
	Lithic density	9.5		101.7		237.8		100.3

Table 3

 3 Debitage composition of the Mirak chipped stone assemblage (L = Layer). Composition de débitage de l'assemblage de pierre écaillée de Mirak (L = couche).

	Tableau 3
	Debitage	L1	%	L2	%	L3	%	Sum
	Bladelets and fragments	4	8.9	21	4.2	4	1.1	29
	Blades and fragments	3	6.7	41	8.2	15	4.1	59
	Flakes and fragments	33	73.3	326	64.9	258	69.7	617
	Chips	5	11.1	114	22.7	93	25.1	212
	Sum	45	100	502	100	370	100	917

Table 4

 4 Tools composition of the Mirak chipped stone assemblage (L = layer). Tableau 4 Composition des outils de l'assemblage en pierre taillée de Mirak (L = couche).phases of human occupation in the Iranian Central Plateau during the late Pleistocene, anchored in a precise geological and chronological framework, in contrast to previous studies, which had relied solely on surface finds. Although caution is needed, from the lithics analysis and the distribution of the finds, at least three main archaeological layers can be identified in Mirak 8. These layers appear to be culturally and chronologically distinct, corresponding to at least three main phases of human occupation. The upper layer (L1) with a maximum age of 28 kya, shows some Upper Paleolithic affinities, although poorly preserved. The intermediate layer (L2) indicates characteristics similar to both Upper and Middle Paleolithic industries that can be seen as an intermediate Paleolithic and dates between 28 ± 2 and 38 ± 2 kya; it appears very original in the context of the Iranian Central Plateau. The lower layer (L3) has clear Middle Paleolithic affinities and dates back to around 47 kya; it would represent a very recent Middle Paleolithic industry compared to what is known in the area. Although further investigations are needed and in progress, Mirak industries point to the complexity of the cultural evolution in a wide area between Zagros and Central Asia.

	Tool type	L1	%	L2	%	L3	%	Sum	%
	Flake tools
	Multiple tools	1	10	5	4.1	13	5.8	19	5.3
	Scraper and notched	0		5		12		17
	Scraper and burin	0		0		1		1
	Scraper and carinated burin	1		0		0		1
	Retouched	3	30	30	24.8	56	24.9	89	25.0
	Burin	2	20	1	0.8	3	1.3	6	1.7
	Simple	0		0		3		3
	Transverse	0		1		0		1
	Carinated	2		0		0		2
	Notch-Denticulated	2	20	17	14	37	16.4	56	15.7
	Scraper	2	20	37	30.6	89	39.6	128	36.0
	Double	1		3		13		17
	Side	1		19		50		70
	End	0		9		6		15
	Convergent	0		6		11		17
	Déjeté	0		0		5		5
	Round	0		0		2		2
	Carinated	0		0		1		1
	Transverse	0		0		1		1
	Point	0	0	9	7.5	6	2.7	15	4.2
	Levallois	0		3		2		5
	Mousterian	0		6		3		9
	Déjeté	0		0		1		1
	Blade Tools	0	0	16	13.2	17	7.5	33	9.3
	Notched/denticulate blade	0		5		3		8	2.2
	Point on blade	0		0		2		2	0.6
	Retouched blade	0		8		7		15	4.2
	Scraper on blade	0		2		4		6	1.7
	Naturally backed knife	0		1		1		2	0.6
	Other tools	0	0	6	5	4	1.8	10	2.8
	Sum	10	100	121	100	225	100	356	100

Acknowledgments

We would like to thank the Research Center for Cultural Heritage and Tourism of Iran (RICHT) and the Iranian Center for Archaeological Research (ICAR) for granting us the permission to conduct the fieldworks at the site. We also would like to express our great gratitude to Mr. Jalil Golshan at the RICHT for his tremendous support during the three seasons of the field missions. We thank the Office of International Affairs at the RICHT for facilitating the process of visa for the non-Iranian partners of the project. We are greatly indebted to Mr. Khajeh Beidokhti, the director of the Iranian Cultural Heritage Organization (ICHO) office in Semnan Province and his colleagues for providing such ideal conditions to conduct the fieldwork. The fieldwork and geological analyses have been cofounded by the office of governor of Semnan (Dr. Khabbaz and his deputy Mr. Zandieh Vakil), the French Ministry of Europe and Foreign Affair (MEAE) and the UMR 7194 CNRS-MNHN-UPVD (Paris); we are very grateful to them. The luminescence dating study was conducted thanks to financial support from the French National Research Agency via the LaS-cArBx Labex (Project number ANR-10-LABX-52). We would also like to thank Patrick August for his help in identification of the fauna remains. Mr. Hassan Rezvani, the discoverer of Mirak, generously allowed us to conduct field research there. We are grateful to Mr. Soroush Hashemi, and Kamran Shojaee at the ICHO office in Semnan for their magnificent logistical support. Dr. Kourosh Mohammadkhani assisted us in geomagnetic fieldwork. Dr. Razieh Lak, Research Deputy at the Geological Survey of Iran and Mr. Habib Poorfaraj, who was in charge of the raw material sourcing, we thank all of them. We are grateful to Mr. Iraj Beheshti at the Iranian Centre for Preservation and Restoration, for his help in identifying the raw material types. We would like to thank Kyle G. Olson for his help in editing the final manuscript. Contributions of co-authors: responsibilities (HVN, GB, AAK), lithics analysis (MiH, MJ, SB), fauna (NS, ZA), geology (GJ, MAK), chronology (GG, MaH), spatial distribution (GB and MiH), design (HVN, GB), and writing of the manuscript (all).

Special thanks go to the field crew in alphabetical order: Alieh Abdollahi, Mahkameh Abolfathi, Laya Alinia, Benoit Chevrier, Nasim Feizi, Mahyar Khademi, Ashghar Nateghi, Mona Oryat, Soroush Razi, Robabeh Sadeghinejad, Yashar Sadeghi, Sanaz Shirvani, Mohammad Javad Shoaee, and Marzieh Zareh Khalili.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j. crpv.2019.02.005.


Hakim Hocini

Aurélie Wiedemann

Fabiola Blengio

Cécile Lefebvre

Minerva Cervantes-Gonzalez

Emile Foucat

Pascaline Tisserand

Mathieu Surenaud

Séverin Coléon

Mélanie Prague

Lydia Guillaumat

Corinne Krief

Craig Fenwick

Cédric Laouénan

Lila Bouadma

Jade Gho

Giuseppe Pantaleo

Rodolphe Thiébaut

Yves Lévy
email: yves.levy@aphp.fr

Laurent Abel

Amal Abrous

Claire Andrejak

François Angoulvant

Delphine Bachelet

Marie Bartoli

Sylvie Behilill

Marine Beluze

Krishna Bhavsar

Anissa Chair

Charlotte Charpentier

Léo Chenard

Cathe- Rine Chirouze

Sandrine Couffin-Cadiergues

Camille Couffignal

Nathalie De Castro

Marie- Pierre Debray

Dominique Deplanque

Diane Descamps

Alpha Diallo

Fernanda Dias

D A Silva

Céline Dorival

Xavier Duval

Philip- Pine Eloy

Vincent Enouf

Hélène Esperou

Marina Esposito-Farese

Manuel Etienne

Aline-Marie Florence

Alexandre Gaymard

Tristan Gigante

Morgane Gilg

François Goehringer

Jérémie Guedj

Ikram Houas

Isabelle Hoffmann

Jean-Sébastien Hulot

Salma Jaafoura

Simon Jamard

Ouifiya Kafif

Antoine Khalil

Nadhem Lafhej

Samira Laribi

Minh Le 15

Quentin Le Hingrat

Soizic Le Mestre

Sophie Letrou

Bruno Lina

Guil- Laume Lingas

Denis Malvy

France Mentré

Hugo Mouquet

Nadège Neant

Christelle Paul

Aurélie Papadopoulos

Ventzislava Petrov- Sanchez

Gilles Peytavin

Valentine Piquard

Olivier Picone

Manuel Rosa-Calatrava

Béné- Dicte Rossignol

Patrick Rossignol

Carine Roy

Marion Schneider

Coralie Tardivon

Jean-François Timsit

Sarah Tubiana

Sylvie Van Der Werf

Visseaux Benoit

Neutrophil Activation and Immune Thrombosis Profiles Persist in Convalescent COVID-19

Keywords: COVID-19 disease, post-acute COVID-19 syndrome, thrombosis

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

Introduction

Following a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, a significant proportion of individuals develop prolonged symptoms, including predominantly fatigue, headache, and upper respiratory and multi-system 1 3 complaints, including fever and gastroenterological symptoms [START_REF] Raveendran | Long COVID: an overview[END_REF]. Several studies have investigated immune and inflammatory dysregulation in acute and severe COVID-19 with the aim of identifying biomarkers of severity or predictive of clinical outcomes. However, the exact mechanisms behind the persistence of symptoms are yet to be identified but they probably result in the COVID-19 sequelae of organ damage, persistence of chronic inflammation, and dysregulation of the immune system [START_REF] Raveendran | Long COVID: an overview[END_REF]. Dysregulation of immune responses, including T-cell lymphopenia and exhaustion [START_REF] Wilk | A single-cell atlas of the peripheral immune response in patients with severe COVID-19[END_REF], and elevated serum levels of pro-inflammatory cytokines or alarmins are associated with a severe prognosis [START_REF] Silvin | Elevated calprotectin and abnormal myeloid cell subsets discriminate severe from mild COVID-19[END_REF][START_REF] Lucas | Longitudinal analyses reveal immunological misfiring in severe COVID-19[END_REF]. In addition, we and others have reported a crucial role for neutrophil activation in the pathology of severe COVID-19 through the upregulation of multiple genes involved in their activation and migration [START_REF] Silvin | Elevated calprotectin and abnormal myeloid cell subsets discriminate severe from mild COVID-19[END_REF][START_REF] Lévy | CD177, a specific marker of neutrophil activation, is associated with coronavirus disease 2019 severity and death[END_REF][START_REF] Veras | SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology[END_REF]. A role for neutrophil extracellular traps (NETosis), both in COVID-19 acute respiratory distress syndrome (ARDS) and thrombotic events, has also been observed [START_REF] Panda | A functionally distinct neutrophil landscape in severe COVID-19 reveals opportunities for adjunctive therapies[END_REF][START_REF] Skendros | Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis[END_REF][START_REF] Uzun | Immune-mediated platelet activation in COVID-19 and vaccine-induced immune thrombotic thrombocytopenia[END_REF]. Concomitantly, severe COVID-19 has also been shown to be associated with altered hematopoiesis, as shown by lymphopenia and an increase in myelopoiesis [START_REF] Netea | Complex immune dysregulation in COVID-19 patients with severe respiratory failure[END_REF][START_REF] Huang | Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China[END_REF]. Single-cell analysis of bone marrow mononuclear cells from severe COVID-19 patients showed an accumulation of immature myeloid and a reduction of lymphoid progenitors, along with the upregulation of transcription factors (TF) regulating the differentiation of hematopoietic stem cells into downstream progenitors [START_REF] Wang | Dysregulated hematopoiesis in bone marrow marks severe COVID-19[END_REF].

Concerning the adaptive immune response to SARS-CoV-2, most COVID-19 patients develop antibodies and T-cell to SARS-CoV-2 antigens [START_REF] Braun | SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19[END_REF][START_REF] Sekine | Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19[END_REF][START_REF] Swadling | T cells in COVID-19 -united in diversity[END_REF]. In the post-acute phase, high titers of specific immunoglobulin G and neutralizing antibodies are detected [START_REF] Ni | Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals[END_REF][START_REF] Cervia | Immunoglobulin signature predicts risk of postacute COVID-19 syndrome[END_REF], as well as specific and polyfunctional T cells with an early differentiated memory phenotype associated with stem-like properties [START_REF] Sekine | Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19[END_REF][START_REF] Arunachalam | Systems biological assessment of immunity to mild versus severe COVID-19 infection in humans[END_REF][START_REF] Kared | SARS-CoV-2-specific CD8+ T cell responses in convalescent COVID-19 individuals[END_REF]. These observations underscore the complexity of the pathophysiological process underlying acute and severe COVID-19. The duration of such dysregulation following the acute phase of the infection is yet to be investigated. In this context, we investigated immune cell phenotypes, SARS-CoV-2 spike specific and neutralizing antibodies, serum biomarkers, and whole blood gene expression profiles in a cohort of hospitalized COVID-19 patients followed from acute phase infection through convalescence 1, 3, and 6 months following discharge from the hospital.

Methods

Participants

We enrolled a subgroup of COVID-19 patients of the prospective French COVID cohort (registered at clinicaltrials.gov NCT04262921). Eligible patients were those who were hospitalized with virologically confirmed COVID-19 by PCR performed on the day of inclusion. The definition criteria of the severity of the disease were stated according to WHO and French National Health Agency guidelines applied to the inclusion of the French National COVID-19 cohort "French cohort" [START_REF] Yazdanpanah | Impact on disease mortality of clinical, biological, and virological characteristics at hospital admission and overtime in COVID-19 patients[END_REF]. Viral loads were quantified by real-time semi-quantitative reverse transcriptase polymerase chain reactions (RT-PCR) using either the Charité WHO protocol (testing the E gene and RdRp) or the Pasteur institute assay (testing the E gene and two other RdRp targets, IP2 and IP4). Convalescent follow-up visits were performed at 2 to 4 weeks after discharge (month 1), and/or month 3, and/or month 6. Among convalescent patients included in the transcriptomic study, 43% and 41% were symptomatic at M3 and M6, respectively. The study was conducted with the understanding and consent of each participant or their surrogate covering the sampling, storage, and use of biological samples. The healthy donors (HD) were sampled before COVID-19 outbreak, their characteristics are shown in Table S3.

Quantification of Serum Biomarkers, Anti-SARS-CoV-2 Spike Antibody, and Pseudo-Neutralization Assay.

We quantified 29 biomarkers in serum samples. A Human XL Cyt Disc Premixed Mag Luminex® Perf Assay Kit (R&D Systems) was used to measure the following 22 biomarkers: CCL2/MCP-1, CCL4/MIP-1β, RANTES/ CCL5, CCL11/Eotaxin, CCL19/MIP-3β, CCL20/MIP-3α, CD40Ligand, Fractalkine/CX3CL1, CXCL10/IP-10, EGF, Flt-3 Ligand, Granzyme B, IL-1ra, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-15, PD-L1/B7-H1, TNF-α, TRAIL/TNFSF10, and VEGF. CD163, ST2, CD14, and LBP were measured with a Human Magnetic Luminex® Assay (R&D Systems). Luminex assays were performed according to the manufacturer's recommendations and read on a Bio-Plex 200 system™ (Bio-Rad). Amphiregulin, FABP2/IFABP, and haptoglobin were measured using Human Quantikine ELISA Kits (R&D Systems) according to the manufacturer's instructions. Anti-SARS-CoV-2 spike immunoglobulin G (IgG) and neutralization levels were determined as already reported [START_REF] Fenwick | A highly potent antibody effective against SARS-CoV-2 variants of concern[END_REF][START_REF] Ferretti | Unbiased screens show CD8(+) T cells of COVID-19 patients recognize shared epitopes in SARS-CoV-2 that largely reside outside the spike protein[END_REF] and detailed in the supplementary data.

RNA Extraction, Library Preparation, and Sequencing.

Gene expression profiling was carried out on the whole blood of 10 HD and of 22, 25, and 18 samples of convalescent patients. Eight, 12, and 9 convalescent patients were sampled only at M1, M3, and M6, respectively. In contrast, 8 patients were sampled both at M1 and M3, 4 patients at M1 and M6, 3 patients at M3 and M6, and only 2 patients at 1 3 M1, M3, and M6. Total RNA was purified using the Tem-pus™ Spin RNA Isolation Kit (ThermoFisher Scientific). RNA was quantified using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific) and quality control performed on a Bioanalyzer (Agilent). Globin mRNA was depleted using the Globinclear Kit (Invitrogen) prior to mRNA library preparation with the TruSeq® Stranded mRNA kit, according to the Illumina protocol. Libraries were sequenced using an Illumina HiSeq 2500 V4 system.

Differential Gene Expression Analysis

Sequencing quality control was performed using Sequence Analysis Viewer and FastQ files were generated on the Illumina BaseSpace Hub. After trimming (QPhred score ≥ 25) with Bowtie 2-2.2.5 software, reads were aligned with the hg19 human reference genome using STAR-2.5.3ar and quantified relative to annotation model hg19-GENCODE Genes-release 19 using Partek E/M (Partek® Flow® software, v10.0). Differential gene expression analysis was performed using the DearSeq package [START_REF] Gauthier | dearseq: a variance component score test for RNA-seq differential analysis that effectively controls the false discovery rate[END_REF]. Differentially expressed genes (DEGs) with adjusted p values ≤ 0.05 and a fold-change in expression ≥ Log2 0.58 were subjected to functional enrichment analysis using Metascape software with the default parameters (https:// metas cape. org) [START_REF] Zhou | Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[END_REF]. Geom_line and geom smooth functions from ggplot2 [25] were used to generate line plots for the gene trend visualization using the LOESS smoothing method, which fits a polynomial surface determined by one or more numerical predictors using local fitting.

Construction of the Protein-Protein Interaction (PPI) Network and Molecular Complex Detection (MCODE)

The interaction network among proteins encoded by the DEGs was established by importing the selected genes into the STRING database v11.5 [START_REF] Szklarczyk | STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets[END_REF], (http:// string-db. org). To remove PPI that were inconsistent from the dataset, we used the standard cut-off of a confident interaction score ≥ 0.4. Cytoscape software version 3.9.0 [START_REF] Shannon | Cytoscape: a software environment for integrated models of biomolecular interaction networks[END_REF] was applied for the genes network visualization. The MCODE [START_REF] Bader | An automated method for finding molecular complexes in large protein interaction networks[END_REF] plug-in of the Cytoscape tool was used to visualize the significant gene modules in each network, by default, with a cut-off k-core = 3 based on the MCODE analysis.

Clustering of Convalescent Severe COVID-19 Patients and Healthy Donors

Hierarchical clustering was used to observe gene expression patterns between subjects and for cluster identification. Feature selection was based on the most highly variable genes (IQR > 0.7). The R package clValid [START_REF] Brock | clValid: an R package for cluster validation[END_REF] was used to identify and validate the cluster algorithm (Figure S7). The "average silhouette width" index from the factoextra R package [START_REF] Kassambara | Extract and Visualize the Results of Multivariate Data Analyses[END_REF] was used to determine the optimal number of clusters. The fviz dend function from the factoextra package was then used to draw the dendrogram.

Statistics

Graphpad Prism software version 8 was used for nonparametric statistics and plots as described in the figure legends. Heatmaps were generated using the function aheatmap from the NMF package in R software, version 4.0.0. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL: https:// www.R-proje ct. org/. Differences in the expression of standardized biomarkers were analyzed using nonparametric Wilcoxon tests, adjusting for multiple testing using the Benjamini & Hochberg correction. A leave-one-out procedure was performed to assess the robustness of the result regarding outlier patients. We performed the same analyses in turn in all but one of the subjects of the dataset and assessed if it impacted which analytes are found to be differently expressed between COVID-19 patients and health donors at each time points.

Results

Participants

We recruited 100 hospitalized COVID-19 patients from the French COVID cohort [START_REF] Yazdanpanah | Impact on disease mortality of clinical, biological, and virological characteristics at hospital admission and overtime in COVID-19 patients[END_REF]. The median (IQR) age was 59 (47-67) years, 66% were male, and 81% were hospitalized in intensive care units (ICUs) during the acute phase of infection. At month (M) 3 and M6 post-hospitalization, 28% and 45% of the patients had three or more persistent symptoms, respectively. Fatigue, abnormal pulmonary auscultation, and dyspnea were the most frequent symptoms. Whole blood samples were obtained longitudinally (2-3 time points) from 33 patients or at a single time point from 69 patients. The demographic and clinical characteristics of patients and the details of the blood samples used in each assay are summarized in Tables S1 andS2.

Overview of Serum Biomarkers and Cellular and Humoral Characteristics of Convalescent COVID-19 Patients

The levels of 21 biomarkers differed significantly in convalescent COVID-19 patients compared to healthy donors (HD, n = 30) (Wilcoxon test adjusted for multiple comparisons) (Fig. 1 and Figure S1) at various time points, of which most persisted until M6 following the acute phase of 1 3 infection. Among the biomarkers showing differences, the levels of pro-inflammatory factors (CCL2, IP-10, and IL-6) and anti-inflammatory cytokines (IL-1-RA and IL-10) were elevated at M1 and M3. The levels of these factors, but not those of CCL2 or IL-10, were still high at M6. Macrophage and endothelial activation marker (MIP-3β, IL-7, VEGF, EGF, and CCL5) levels were also elevated up through M6. Accordingly, elevated levels of soluble CD163 (sCD163), a specific marker of monocyte/macrophage activation, and chemotaxis and hematopoietic cytokines (CCL11, fractalkine, and Flt-3L) also persisted up to 6 months. The levels of lipopolysaccharide-binding protein (LBP) and intestinal fatty acid-binding protein (iFABP), a marker of gut epithelial damage, were also significantly elevated in convalescent COVID-19 patients at M3. Consistent with this finding, the levels of markers of intestinal epithelial regeneration (EGF, amphiregulin AREG, and haptoglobin), soluble ST2, and sCD14, a marker of intestinal translocation, were elevated at various time points up to M6 (Fig. 1 and Figure S1E). Using a leave-one-out validation, we demonstrated that these findings are robust to outliers patients.

We also performed phenotypic analysis of the blood immune cells from the convalescent COVID-19 patients. There was a significant reduction in the frequency of total CD3 + T cells up to 3 months after the acute phase relative to HD (P = 0.099 and P = 0.0375 at M1 and M3, respectively), whereas the number of central memory CD4 + T cells was elevated at M1 and M3 (P = 0.0073 and P = 0.0143, respectively). The frequency of naïve CD8 + T cells was reduced at M1, whereas the frequency of effector memory CD8 + T cells was elevated at M1 and M3 and they expressed an activated cell phenotype (CD38 + HLA-DR + ) until M1 (P < 0.0001 relative to HD) (Figure S2A).

The mean fluorescence intensity (MFI) of anti-SARS-CoV-2 spike antibody for convalescent COVID-19 patients was 368.8 (252.2-610.8) fold higher at M1 than that for a pool of HD, which then decreased by M3 (201.7 (142.7-342.1)) and M6 (176.5 (135-258)) (P < 0.0001, for all time points) (Figure S2B). Similarly, the percentage of inhibition of these antibodies was 68.6 (58-84) at M1, which decreased to 37.6 (29-57) and 41.9 (33-50) by M3 and M6, respectively (Figure S2B).

Changes in Whole Blood Gene Expression in Convalescent COVID-19 Patients Relative to HD

To characterize the transcriptomic changes that occur in convalescent COVID-19 patients, we investigated DEGs between samples collected at M1 (n = 22), M3 (n = 25), and M6 (n = 18) and HD (n = 10). We identified 1754, 864, and 871 DEGs at M1, M3, and M6, respectively (Fig, 2A). The DEGs at M1 involved pathways of myeloid cell differentiation, neutrophil degranulation, transcriptional regulation of granulocytes, and platelet activation (Fig. 2B). Among the top upregulated genes, some are involved in myeloid lineage and required for granulocyte formation (CEBPA and GATA1). Others are involved in erythrocyte and platelet differentiation and activation, such as ALAS2, GP1BB, GP6, GP9, VWF, GP1BA, and HBQ1, which is normally found in human fetal erythroid tissue. Interestingly, the top downregulated gene, HBB, is one of the subunits of adult hemoglobin (Fig. 2C). Aside from these genes, at M1 after hospital discharge, we also observed dysregulation of genes related to the myeloid lineage (Figure S3A), which persisted until M6 (Figure S3B). PPI analysis (Figure S3C) identified the main network, which involved the upregulation of TF genes associated with myeloid and erythrocyte cell differentiation (CEBPA, CEBPE, GATA1, GATA2, and KLF1), as well as genes belonging to GP1b-IX-V activation (GP1BB, GP9, and GP1BA). Of note, the GP1b-IX-V complex is a platelet receptor that mediates the initial interaction with subendothelial vWF which levels were elevated in COVID-19 patients [31]. The involvement of platelet activation was confirmed, as shown by the dysregulation of multiple genes related to platelets (Figure S4A). Among these genes, we observed two main clusters. The first cluster (C1) consisted of the genes downregulated at M1 relative to HD (PRKACB, ENPP4, HBB, and FZD6) (Figure S4A), which are involved in blood coagulation. The second cluster (C2) included genes upregulated at M1 that are mainly involved in F9 and GP1b-IX-V activation, platelet adhesion and aggregation, and plug formation. All these genes were dysregulated at M1 relative to HD and did not return to HD level at M6 (Figure S4B). The PPI results confirm the key role of the blood coagulation and platelet activation in the dysregulated mechanisms observed at day 1 (Figure S4C).

We have previously reported that changes in neutrophil activation genes are predictive of severe disease in COVID-19 [START_REF] Lévy | CD177, a specific marker of neutrophil activation, is associated with coronavirus disease 2019 severity and death[END_REF]. Thus, we took the opportunity of the longitudinal follow-up of cohort of convalescent COVID-19 patients to follow neutrophil gene pathways. We observed modified expression of many genes associated with neutrophil activation (Figure S5A). Two main clusters of DEGs were identified that exhibited different dynamics. One cluster decreased expression at M1 and remained downregulated, despite a tendency to increase by M6. By contrast, the expression of genes in the second cluster was increased at M1 and remained higher up to M6 (Figure S5B). This cluster included genes of neutrophil degranulation (S100A9, DEFA1B, A1BG, MMP9, PPBP, SIGLEC5, and SIGLEC9). Network analysis of the neutrophil genes identified major pathways concerning neutrophil activation/degranulation and complement C3b/C4b receptor 1 and C5a receptor 1 (C5AR1) (Figure S5C).

We next performed MCODE analysis using all DEGs observed between COVID-19 patients at M1 and the HD to determine the major mechanisms behind the dysregulation observed in convalescent COVID-19 patients. This approach allowed the identification of three highly connected networks (Fig. 3). Network 1 is distinguished by the upregulation of genes involved in the RUNX1 signaling in the regulation of megakaryocyte and erythroid differentiation, and platelet function (GATA1, SPI1, ZFPM1, and TAL1) [START_REF] Vagapova | The role of TAL1 in hematopoiesis and leukemogenesis[END_REF][START_REF] Kuvardina | RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation[END_REF][START_REF] Elagib | RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation[END_REF][START_REF] Mancini | FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors[END_REF] (Fig. 3A). Importantly, this network included the upregulation of GP1BA, a receptor for vWF that leads to platelet adhesion and activation. In parallel, in network 2 (Fig. 3B), we observed the upregulation of several genes involved in platelet pro-coagulation activity and subsequent thrombin and fibrin formation, such as GP1BB, VWF, PF4, ITGB3, GP6, and GP9, which functions as the vWF receptor and mediates vWF-dependent platelet adhesion to blood vessels [START_REF] Denorme | von Willebrand factor and platelet glycoprotein Ib: a thromboinflammatory axis in stroke[END_REF][START_REF] Levi | COVID-19 coagulopathy vs disseminated intravascular coagulation[END_REF]. Concomitantly, network 3 (Fig. 3C) was enriched for the expression of genes of the hemoglobin complex and glycophorin (HBB, KLF1, SLC4A1, AHSP, RHAG, and SPTA1). Despite the heterogeneity of patients, as illustrated in the heatmaps of individual gene expression (Figs. 3 D-F), these results show an interaction between platelets and neutrophil activation pathways that persists to M3 and M6. Moreover, the dynamics of these changes did not show a return to HD levels at the later follow-up after discharge (Figs. 3 G-I).

In other hand, DEGs (314 genes) shared at M1 and M3 relative to HD (Figure S6A) are mainly involved in protein metabolism (Figure S6B). DEGs specific to convalescent patients at M6 (157 genes) and M3 (50 genes) involved pathways of aminophospholipid translation, heme signaling, cell morphogenesis, and inflammatory response (Figure S6C) and glycerolipid metabolic processes (Figure S6D). We noted significant enrichment of genes involved in erythrocyte differentiation and granulopoiesis (Figure S6E) at M1 and M6 (Figure S6F). These DEGs showed the same kinetics at M1 and M6 (Figure S6G).

Characterization of a "Core Signature" of Gene Expression in Convalescent COVID-19 Patients

We analyzed pathways involved in the 477 DEGs shared between M1, M3, and M6 relative to HD (Fig. 2A) to identify a "core signature" of convalescent COVID-19 patients throughout the 6 months of follow-up. The main pathways involved are myeloid cell differentiation, platelet adhesion and activation, transcriptional regulation by RUNX1, and granulopoiesis (Fig. 4A). Analysis of the most connected network identified genes that play a critical role in collageninduced platelet aggregation and thrombus formation (GP6 and GP9) and in the GPIb-V-IX system (GP1BB, GP1BA). A second cluster consisted of upregulated genes regulated by RUNX1 and involved in megakaryocyte differentiation, platelet function, and the transcriptional regulation of granulopoiesis (CEBPA and ZFPM1) (Figs. 4B andC). The expression of all these genes was significantly elevated at M1, M3, and M6 of convalescence (Figs. 4D andE) and did not return HD levels by M6 (Figs. 4F andG).

Gene Expression Profile Associated with Thrombosis in Convalescent Severe COVID-19 Patients

We further characterized the gene expression profile that could be associated with thrombosis by performing a clustering analysis of HD and samples from convalescent patients at M1, M3, and M6. The samples were grouped into two main clusters (Fig. 5A). Cluster C1 included HD and convalescent COVID-19 patients who did not experience thrombotic events, showing very similar transcriptome profiles. Cluster C2 included a mixture of convalescent patients who experienced thrombosis and those who did not. This cluster likely corresponds to patients with a high risk of thrombosis. Surprisingly, this cluster included 3 HD who may be at risk of developing thrombosis. DEGs analyses of the convalescent patients in cluster 1 and those who experienced thrombosis in cluster C2 showed 1333 DEGs that were associated with pathways of the innate immune system, neutrophil degranulation, hemopoiesis, platelet activation, and blood coagulation (Fig. 5B). MCODE analysis resulted in four main clusters. The first one (Fig. 5C) included genes involved in neutrophil and complement activation (S100A9, ARG1, ELANE, C5AR1, and FCGR3B), and in the hemoglobin and erythrocyte development (ALAS2, GATA2, HBB, HBD, HBG1, HBA1HBQ1, and AHSP). The second cluster (Fig. 5D) corresponded to neutrophil degranulation, with an upregulation of S100A8, S100A12, SPI1, TYROBP, FGR, FCGR1A, and FCGR2A. Cluster 3 (Fig. 5E) contained mostly genes involved in reactive oxygen and nitrogen species and thrombin signaling. The last cluster (Fig. 5F) contained upregulated genes belonging to the stress response, mainly mediated by neutrophils (DEFA4, DEFA1B, DEFA3, DEFA1, MPO, LTF, MMP8, and MMP9) and to coagulation (GP6, GP9, GP1BA, PF4, CLEC1B, and THBD). Overall, convalescent individuals, even those who were asymptomatic, showed persistent changes in the expression of genes involved in inflammation and a status of being prone to thrombosis that lasted for up to 6 months following acute disease.

Discussion

Here, we report persistent severe abnormalities in the blood of convalescent COVID-19 patients. An abnormal immune cell phenotype, elevated serum markers of inflammation, endothelial activation, and intestinal translocation coexist with anti-inflammatory and epithelial repair markers. Whole blood gene expression analyses showed marked changes in the expression of genes involved in several pathogenic pathways involving hematopoiesis, platelet, erythrocyte, and neutrophil activation. The altered gene expression did not return to the profile of HD after 6 months of follow-up. Another original finding from our study is the identification of a "core signature" of convalescent COVID-19 patients that is conserved for up to 6 months after hospital discharge, suggesting the persistence of pathophysiological processes, not only in patients who are still symptomatic but also those without any "overt" sequelae. Finally, we identified a gene expression profile associated with thrombosis that occurred in a subgroup of convalescent COVID-19 patients.

We first confirmed a decrease in the frequency of peripheral T lymphocytes and a relative increase in the frequency of central memory CD4 + and activated effector CD8 + T cells until M3 [START_REF] Fathi | Lymphopenia in COVID-19: therapeutic opportunities[END_REF]. Concomitantly, serum cytokine profiling showed a balance between pro-inflammatory factors and anti-inflammatory cytokines at M1 and M3. Remarkably, the levels of markers of monocyte/macrophage and endothelial activation, chemotaxis and hematopoietic cytokines, were still elevated several months after acute infection.

Longitudinal follow-up showed changes in blood gene expression, with marked dysregulation of the activation, signaling, and aggregation of platelet homeostasis, transcriptional regulation of granulopoiesis, and neutrophil degranulation. Overall, dysregulation of these pathways was common throughout the convalescent COVID-19 patient population, emphasizing a "core signature," with upregulation of the RUNX1 pathway, which regulates the expression of many dysregulated genes [START_REF] Kuvardina | RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation[END_REF][START_REF] Kellaway | Molecular basis of hematological disease caused by inherited or acquired RUNX1 mutations[END_REF]. Of note, up to 78% of these genes exhibit binding sites for this factor, highlighting the important role of RUNX1 in the physiopathology of COVID-19 [START_REF] O'hare | Targeting runt-related transcription factor 1 prevents pulmonary fibrosis and reduces expression of severe acute respiratory syndrome Coronavirus 2 host mediators[END_REF].

Clinically, the persistence of pathophysiological processes is illustrated by "emergency hematopoiesis" well beyond clinical recovery, as illustrated by the upregulation of key genes involved in the hematopoietic cell differentiation of granulopoiesis, erythroid and megakaryocytic cell, coagulation, platelet activation, and aggregation. These results extend previous observations of abnormal hematopoiesis during the convalescent stage of severe and acute COVID-19 patients [START_REF] Wang | Dysregulated hematopoiesis in bone marrow marks severe COVID-19[END_REF][START_REF] Lippi | Thrombocytopenia is associated with severe coronavirus disease 2019 (COVID-19) infections: a meta-analysis[END_REF].

Coagulopathy is an important cause of morbidity and mortality among patients with COVID-19 [START_REF] Al-Samkari | COVID-19 and coagulation: bleeding and thrombotic manifestations of SARS-CoV-2 infection[END_REF][START_REF] Bongiovanni | SARS-CoV-2 infection is associated with a pro-thrombotic platelet phenotype[END_REF] and SARS-CoV-2 induces endothelial disruption and vascular thrombosis in histopathologic sections of lungs from autopsies of both humans and rhesus macaques infected with SARS-CoV-2 [START_REF] Aid | Vascular disease and thrombosis in SARS-CoV-2-infected rhesus macaques[END_REF]. Histological analysis of pulmonary vessels in acute COVID-19 has shown widespread thrombosis with microangiopathy [START_REF] Ackermann | Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19[END_REF][START_REF] Nicolai | Immunothrombotic dysregulation in COVID-19 pneumonia is associated with respiratory failure and coagulopathy[END_REF]. Relative to healthy controls, we show persistent upregulation of genes involved in platelet pro-coagulation activity and subsequent thrombin and fibrin formation, as well as genes that mediate vWF-dependent platelet adhesion to blood vessels. Consistent with these observations, a potential role of vWF and complement activation in COVID-19-associated coagulopathy has been proposed [START_REF] Goshua | Endotheliopathy in COVID-19-associated coagulopathy: evidence from a single-centre, cross-sectional study[END_REF]. Our results provide a pathophysiological explanation for the higher incident risk of deep vein thrombosis, pulmonary embolism, bleeding, and ischemic cardiovascular events among convalescent COVID-19 patients [START_REF] Katsoularis | Risks of deep vein thrombosis, pulmonary embolism, and bleeding after covid-19: nationwide self-controlled cases series and matched cohort study[END_REF]. Interestingly, a high relative incidence of vascular events was observed soon after COVID-19 diagnosis that declined rapidly but incidence remains elevated up to 7 months after COVID-19 diagnosis [START_REF] Knight | Association of COVID-19 with major arterial and venous thrombotic diseases: a population-wide cohort study of 48 million adults in England and Wales[END_REF]. Our study extended this observation by confirming a persistent risk of vascular events and by providing a pathophysiological profile of the convalescent patients. These observations raise the question of the maintenance of prevention measures to avoid thromboembolism events [START_REF] Connors | COVID-19 and its implications for thrombosis and anticoagulation[END_REF].

Elevated NET markers have been reported in the serum of acute severe COVID-19 patients, associated with inflammatory cytokine release, coagulopathy, and respiratory failure [START_REF] Zuo | Neutrophil extracellular traps in COVID-19[END_REF]. Here, we demonstrate that 6 months after hospital discharge, convalescent patients still exhibit a significant increase in the expression of genes of neutrophil degranulation, NET and alarmins [START_REF] Mócsai | Integrin signaling in neutrophils and macrophages uses adaptors containing immunoreceptor tyrosine-based activation motifs[END_REF], illustrating the role of neutrophils in blood coagulation and thrombosis, probably via interaction with both the injured endothelium and fibrinogen [START_REF] Shi | Neutrophils can promote clotting via FXI and impact clot structure via neutrophil extracellular traps in a distinctive manner in vitro[END_REF][START_REF] Kapoor | The role of neutrophils in thrombosis[END_REF][START_REF] Iliadi | The emerging role of neutrophils in the pathogenesis of thrombosis in COVID-19[END_REF]. Overall, our data underscore the central role of the trio of platelets, neutrophils, and erythrocytes in the disturbances observed in convalescent severe COVID-19 patients.

We sought to characterize the gene expression profile associated with thrombotic events by clustering analysis. Despite a degree of heterogeneity, all convalescent patients who experienced thrombosis were grouped within the same cluster. This cluster was highly dominated by dysregulation of the RUNX1 TF, neutrophil degranulation, and thrombin signaling pathways. Convalescent COVID-19 patients who experienced thrombosis also showed upregulation of hemoglobin complex and erythrocyte development genes, as well as genes of platelet activation and blood coagulation. It is now admitted that infections increase the risk of thrombosis independently of the risk factors for thromboembolic diseases [START_REF] Beristain-Covarrubias | Understanding infectioninduced thrombosis: lessons learned from animal models[END_REF]. Beside SARS-CoV-2, influenza and cytomegalovirus can also enhance the risk of thrombosis.

The key factor that probably underlies the risk of thrombosis is the level of inflammation induced by the infection, which can activate platelets through different receptors, triggering aggregation and thrombi formation. Interestingly, the hospitalized COVID-19 patients have significant higher thrombosis incidence relative to hospitalized patients with influenza [START_REF] Stals | Risk of thrombotic complications in influenza versus COVID-19 hospitalized patients[END_REF]. This difference is probably due to a higher pro-coagulant status induced in COVID-19 subsequent to the activation of both platelets, neutrophils and erythrocytes as confirmed in our study.

The reasons for the persistence of dysregulation after clinical recovery are not obvious but suggest a continuous pathophysiological process both in patients who are still symptomatic and those without any "overt" clinical sequelae [START_REF] Liu | Prolonged virus shedding even after seroconversion in a patient with COVID-19[END_REF][START_REF] Van Kampen | Duration and key determinants of infectious virus shedding in hospitalized patients with coronavirus disease-2019 (COVID-19)[END_REF][START_REF] Folgueira | Prolonged SARS-CoV-2 cell culture replication in respiratory samples from patients with severe COVID-19[END_REF][START_REF] Havervall | Symptoms and functional impairment assessed 8 months after mild COVID-19 among health care workers[END_REF][START_REF] Phetsouphanh | Immunological dysfunction persists for 8 months following initial mild-to-moderate SARS-CoV-2 infection[END_REF]. The mechanism behind this phenomenon is still unknown, although several hypotheses have been suggested, such as viral reactivation and/or persistent viral reservoirs or antigens [START_REF] Alhusseini | Persistence of SARS-CoV-2: a new paradigm of COVID-19 management[END_REF][START_REF] Patterson | Persistence of SARS CoV-2 S1 protein in CD16+ monocytes in post-acute sequelae of COVID-19 (PASC) up to 15 months post-infection[END_REF]. In addition, we show high expression of markers that suggest a lack of integrity in the intestinal barrier and ongoing tissue repair in convalescent COVID-19 patients, suggesting the persistence of a pathological phenomenon at the mucosal level [START_REF] Vanella | Gastrointestinal mucosal damage in patients with COVID-19 undergoing endoscopy: an international multicentre study[END_REF].

Our study had several limitations. Because of the limitations in sampling, all assays were not performed in the global cohort and a limited number of patients were sampled at M1, M3, and M6. Furthermore, the convalescent patients were not perfectly matched in age and gender with healthy donors. Indeed, convalescent patients and healthy donors were of 66% and 100% males with mean age of 59 and 35 years, respectively. Another limitation of this study is the possible heterogeneity with regard to the time from initial infection by SARS-Cov-2, the duration of hospitalization, and the severity of the disease. In this study, we followed mostly hospital patients who experienced severe COVID-19. It will be informative to investigate whether certain immune and transcriptomic signatures are reproducibly identified in moderate COVID-19 patients.

In conclusion, we describe a complex pathophysiological process in which both severe mediators of tissue injury and 1 3 thrombotic complications and mechanisms for the control of a hyperinflammatory state coexist in patients who are still symptomatic (50% of our cohort) and those without overt symptoms. Our results suggest the persistence of activation of neutrophils and disturbance of the coagulation pathway in many convalescent COVID-19 patients. Hence, convalescent sever COVID-19 patients who experienced thrombotic events during the acute phase of the disease show an upregulation of genes involved in neutrophil activation, platelet, and blood coagulation. The lack of restoration of gene expression to a normal profile after up to 6 months of follow-up indicates the need to carefully extend their clinical follow-up and propose preventive measures.

Fig. 1

 1 Fig. 1 Heatmap of standardized biomarker expression in the serum of convalescent COVID-19 patients at M1, M3, and M6 post-infection. The colors represent standardized expression values centered on the mean, with variance equal to 1. Biomarker hierarchical clustering was computed using the Euclidean distance and Ward's method. HD,

Fig. 2

 2 Fig. 2 Differentially expressed genes between convalescent patients at M1, M3, and M6 relative to healthy donors (HD). A Venn diagram of the number of DEGs between convalescent patients at M1, M3, and M6 relative to HD. B Pathways associated with DEGs

Fig. 3

 3 Fig. 3 The main molecular complexes associated with the DEGs between convalescent patients at M1 relative to HD and their evolution over the time. A-C MCODE 1, 2, and 3, respectively, and the main associated pathways. The spheres are colored depending on the gene fold change (Log2FC), as depicted in the figure. Pathways asso-

Fig. 4

 4 Fig. 4 Differentially expressed genes shared between the comparisons of convalescent patients at M1, M3, and M6 to HD. A Pathways associated with 477 DEGs shared between convalescent patients at M1, M3, and M6 relative to HD. B, C MCODE 1 and 2 of the 477 DEGs and their main pathways. The spheres are colored depending

Fig. 5

 5 Fig. 5 Clustering of HD and convalescent COVID-19 patient samples recovered at M1, M3, and M6 and gene expression profile associated with thrombosis in convalescent COVID-19 patients. A Clustering of HD and convalescent COVID-19 patient samples recovered at M1, M3, and M6. B Pathways associated with DEGs between convalescent patients who experienced thrombosis in cluster C2 and those who did not experience thrombosis and classified close to HD

Acknowledgements

We thank the patients who donated their blood. We also thank F. Mentre, S. Tubiana, the French COVID cohort, and REACTing (REsearch & ACtion emergING infectious diseases) for cohort management. We thank the scientific advisory board of the French COVID-19 cohort, composed of Dominique Costagliola, Astrid Vabret, Hervé Raoul, and Laurence Weiss. Members of the French COVID study group and their affiliations.

Supplementary Information

The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s10875-023-01459-x. Data Availability RNA-sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) repository with the accession codes GSE227116. The hg19 human reference genome can be found in the NCBI database.

Author Contribution

Declarations

Ethical Approval The study protocol was approved by the French Ethics Committee CPP-Ile-de-France VI (ID RCB: 2020-A00256-33) on February

Consent for Publication

The authors consent to publish the content of this original report.

Conflict of Interest The authors declare no competing interests.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Charles Savoldelli

Mohamed Syahmi Shaik

Yannick Tillier

Yannick Tillier Predicting

Mohamed Syahmi

Predicting The Risk of Fracture of Osteosynthesis Plates

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

Introduction

The case of a partially edentulous patient who underwent orthognathic surgery is presented. A fracture of a right double plate was observed several weeks after surgery (Fig. 1, right). The objective of this work is to demonstrate whether it is possible to predict the risk of postoperative fracture of osteosynthesis plates using finite element (FE) modeling.

Materials and Methods

Fortunately, a CT scan of the patient had been performed prior to surgery. The 3D images were sent to Materialise Medical (Leuven, Belgium), for image segmentation and surgical planning. This allowed them to provide mesh files of the patient's facial structure and of the custom osteosynthesis plates used for the surgery. All meshed objects (ramus, mandible, maxilla, teeth, plates) were imported into the FE model Forge® NxT 3.2 (Transvalor, Sophia Antipolis, France). They were placed in contact with their neighbors and were considered as linear elastic and compressible materials in first approximation. The material data were taken from the literature [1].

Two different methods to get the desired jaw clenching movement for the simulations were used: (i) a set of muscle forces (masseter, temporalis and medial pterygoid) gradually increased up to 200 N along x, y and z axes respectively, resulting in an upward displacement of the mandible [2] and (ii) an upward displacement of a translation tool under the mandible. The calculations were arbitrarily stopped after a maximum imposed displacement of 0.016 mm, the forces becoming too high beyond that (teeth are already in contact at the beginning).

Two kinds of boundary conditions to simulate plate fixation (representative of a consolidated state after healing) were evaluated: (i) with screws inside drilled bones (perfect contact) or (ii) without screws through a condition of bilateral sticking contact at the interface between the plate and the bone (Fig. 1, left).

Results

Not considering screws in the simulation does not allow the computations to converge. Screws tend to stabilize the system while increasing the contact surfaces and thus the computation times. Only the cases with screws were then analyzed. To push under the mandible allows the calculation to converge much faster than using a set of muscular forces, while also being much easier to define. The results show that the von Mises stress levels are higher at the plates, differently on the left and on right sides, with the highest values on the right plate (22 MPa vs 13 MPa), i.e. the side where the patient is partially edentulous, and furthermore in the area where the plate broke after surgery.

Discussion and Conclusions

The surgical planning tools currently used in maxillofacial surgery are mainly based on geometric data. This study shows that the mechanical aspects should not be neglected.

Figure 1 :

 1 Figure 1: Finite element model (left) and von Mises stress distribution in the plate failure area (right).