Romie Tignat-Perrier

Aurélien Dommergue

Alban Thollot

Olivier Magand

Pierre Amato

Muriel Joly

Karine Sellegri

Timothy M Vogel

Catherine Larose

Seasonal shift in airborne microbial communities linked to land use

Keywords: atmospheric microorganisms, bioaerosols, planetary boundary layer, amplicon sequencing, biosphere-atmosphere interactions

Highlights

-Airborne microbial communities showed a seasonal shift at the puy de Dôme elevated site -Dominant microbial taxa showed different trends throughout the year -Summer results in higher concentrations of plant-associated microbes in the air -Winter results in higher concentrations of soil and dead material-associated microbes -Seasonal changes in the underlying ecosystems likely drive microbial seasonal shift Abstract 2 Microorganisms are ubiquitous in the atmosphere. Global investigations on the geographical and temporal distribution of airborne microbial communities are critical for identifying the sources and the factors shaping airborne communities. At mid-latitude sites, a seasonal shift in both the concentration and diversity of airborne microbial communities has been systematically observed in the planetary boundary layer. While the factors suspected of affecting this seasonal change were hypothesized (e.g., changes in the surface conditions, meteorological parameters and global air circulation), our understanding on how these factors influence the temporal variation of airborne microbial communities, especially at the microbial taxon level, remains limited. Here, we investigated the distribution of both airborne bacterial and fungal communities on a weekly basis over more than one year at the midlatitude and continental site of puy de Dôme (France; +1465 m altitude above sea level). The seasonal shift in microbial community structure was likely correlated to the seasonal changes in the characteristics of puy de Dôme's landscape (croplands and natural vegetation). The airborne microbial taxa that were the most affected by seasonal changes trended differently throughout the seasons in relation with their trophic mode. In addition, the windy and variable local meteorological conditions found at puy de Dôme were likely responsible for the intraseasonal variability observed in the composition of airborne microbial communities.

Introduction

Thousands to millions of diverse microbial cells per cubic meter of air are transported among aerosols with their diversity shown to depend on geographic location 1 and time of year [START_REF] Uetake | Seasonal changes of airborne bacterial communities over Tokyo and influence of local meteorology[END_REF] . These microorganisms might be active, since some airborne microbial isolates were shown in laboratory studies to sustain metabolic activity using organic acids found in the atmosphere [START_REF] Amato | A fate for organic acids, formaldehyde and methanol in cloud water: their biotransformation by micro-organisms[END_REF][START_REF] Vaïtilingom | Contribution of Microbial Activity to Carbon Chemistry in Clouds[END_REF][START_REF] Ariya | Microbiological degradation of atmospheric organic compounds[END_REF] .

Understanding the global distribution of airborne microbial communities is critical for determining how airborne microorganisms might influence atmospheric chemistry [START_REF] Vaïtilingom | Contribution of Microbial Activity to Carbon Chemistry in Clouds[END_REF] , meteorological processes such as cloud and precipitation formation [START_REF] Pöschl | Rainforest Aerosols as Biogenic Nuclei of Clouds and Precipitation in the Amazon[END_REF] , as well as human and crop health [START_REF] Brown | Aerial dispersal of pathogens on the global and continental scales and its impact on plant disease[END_REF] . Recent large geographical and spatial investigations highlighted the major contribution of the local landscapes and local environmental factors in the observed distribution of airborne microbial communities in the planetary boundary layer [START_REF] Tignat-Perrier | Global airborne microbial communities controlled by surrounding landscapes and wind conditions[END_REF][START_REF] Mhuireach | Spatiotemporal Controls on the Urban Aerobiome[END_REF] . The composition of airborne microbial communities is closely related to the nature of the surrounding landscapes (ocean, agricultural soil, forest etc.) from which local meteorology (especially wind direction and speed) controls microbial cell emission rates [START_REF] Tignat-Perrier | Global airborne microbial communities controlled by surrounding landscapes and wind conditions[END_REF] . Studies on airborne microbial communities at mid-latitude sites (aerosol-, cloud water-and precipitationassociated microorganisms) reported seasonal changes in both microbial biomass and biodiversity [START_REF] Uetake | Seasonal changes of airborne bacterial communities over Tokyo and influence of local meteorology[END_REF][START_REF] Innocente | Influence of seasonality, air mass origin and particulate matter chemical composition on airborne bacterial community structure in the Po Valley, Italy[END_REF][START_REF] Bowers | Seasonal variability in airborne bacterial communities at a high-elevation site[END_REF][START_REF] Bowers | Seasonal variability in bacterial and fungal diversity of the near-surface atmosphere[END_REF][START_REF] Franzetti | Seasonal variability of bacteria in fine and coarse urban air particulate matter[END_REF][START_REF] Cáliz | A long-term survey unveils strong seasonal patterns in the airborne microbiome coupled to general and regional atmospheric circulations[END_REF] . The seasonal variability was associated to changes in surface conditions [START_REF] Bowers | Seasonal variability in airborne bacterial communities at a high-elevation site[END_REF][START_REF] Bowers | Seasonal variability in bacterial and fungal diversity of the near-surface atmosphere[END_REF] , meteorological conditions [START_REF] Uetake | Seasonal changes of airborne bacterial communities over Tokyo and influence of local meteorology[END_REF][START_REF] Innocente | Influence of seasonality, air mass origin and particulate matter chemical composition on airborne bacterial community structure in the Po Valley, Italy[END_REF] and/or changes in the global air circulation [START_REF] Innocente | Influence of seasonality, air mass origin and particulate matter chemical composition on airborne bacterial community structure in the Po Valley, Italy[END_REF][START_REF] Cáliz | A long-term survey unveils strong seasonal patterns in the airborne microbiome coupled to general and regional atmospheric circulations[END_REF] . Yet, our understanding on how these potential factors impact airborne microbial community composition, and more specifically the microbial taxa individually, remains limited. Here, we investigated the distribution of airborne microbial communities and specific microbial taxa at the mid-latitude and continental site of puy de Dôme (France; +1465 m altitude above sea level). We monitored the diversity and abundance of bacterial and fungal communities in the troposphere on a weekly basis for more than a year (June 2016 to August 2017). These microbial community metrics were evaluated in relation to the local meteorology and particulate matter chemical composition, and puy de Dôme local landscape was evaluated based on the MODIS satellite images. While a number of studies focusing on microorganisms in clouds has been carried out at puy de Dôme [START_REF] Amato | A fate for organic acids, formaldehyde and methanol in cloud water: their biotransformation by micro-organisms[END_REF][START_REF] Vaïtilingom | Contribution of Microbial Activity to Carbon Chemistry in Clouds[END_REF][START_REF] Amato | Metatranscriptomic exploration of microbial functioning in clouds[END_REF][START_REF] Amato | Active microorganisms thrive among extremely diverse communities in cloud water[END_REF][START_REF] Amato | Survival and ice nucleation activity of bacteria as aerosols in a cloud simulation chamber[END_REF][START_REF] Vaïtilingom | Potential impact of microbial activity on the oxidant capacity and organic carbon budget in clouds[END_REF] , no investigation was conducted in the dry troposphere and at such a high temporal resolution. Puy de Dôme is surrounded mainly by croplands and vegetation (i.e. > 80% of the surrounding landscapes in a perimeter of 50 km) whose surface characteristics change drastically over the four different seasons (summer, autumn, winter and spring). These seasonal changes in landscape were related to the temporal variability of airborne microbial community composition at the microbial taxon level.

Material and Methods

Sites and Sampling

A size selective high volume air sampler installed at the puy de Dôme (PDD) meteorological station terrace was used to collect particulate matter on quartz fiber filters every week from June 2016 to August 2017 (Table S1). The sampler was equipped with a PM10 size-selective inlet in order to collect particulate matter smaller than 10 µm (PM10) and sampling was done as presented in Dommergue et al. (2019) [START_REF] Dommergue | Methods to investigate the global atmospheric microbiome[END_REF] . Overall, the dataset was composed of fifty-three samples with an average normalized collected volume of 9100 m 3 (Table S1). Quartz fiber filters were heated to 500°C for 8 hours to remove traces of organic carbon including DNA.

All the material including the filter holders, aluminium foils and plastic bags in which the filters were transported were sterilized using UV radiation as detailed in Dommergue et al., (2019) [START_REF] Dommergue | Methods to investigate the global atmospheric microbiome[END_REF] . A series of field and transportation blank filters were carried out to monitor the quality of the sampling protocol as presented in Dommergue et al. (2019) [START_REF] Dommergue | Methods to investigate the global atmospheric microbiome[END_REF] . PDD is a midaltitude (+ 1465 m) site surrounded by croplands, an urban area (Clermont-Ferrand) and forests within a 50 km perimeter (Fig. 1). Monthly NASA satellite images of puy de Dôme surrounding surfaces (https://wvs.earthdata.nasa.gov/) are shown in Fig. S1. The Atlantic coast and Mediterranean Sea are at around 320 km and 240 km from PDD, respectively.

DNA extraction

We extracted DNA from 3 punches (diameter of one punch: 38 mm) from the quartz fiber filters using the DNeasy PowerWater kit with some modifications as detailed in Dommergue et al. (2019) [START_REF] Dommergue | Methods to investigate the global atmospheric microbiome[END_REF] . During cell lysis, an one hour heating step at 65°C followed by a 10-min vortex treatment at maximum speed and a centrifugation using a syringe to separate the filter debris from the lysate were added to the DNeasy PowerWater DNA extraction protocol [START_REF] Dommergue | Methods to investigate the global atmospheric microbiome[END_REF] .

DNA concentration was measured using the High Sensitive Qubit Fluorometric Quantification (Thermo Fisher Scientific) then stored at -20°C.

Real-Time qPCR analyses

The bacterial cell concentration was approximated by the number of 16S rRNA gene copies per cubic meter of air and the fungal cell concentration was approximated by the number of 18S rRNA gene copies per cubic meter of air. Primers and methodology are presented in Tignat-Perrier et al. (2019) [START_REF] Tignat-Perrier | Global airborne microbial communities controlled by surrounding landscapes and wind conditions[END_REF] . [START_REF] Tignat-Perrier | Global airborne microbial communities controlled by surrounding landscapes and wind conditions[END_REF] . The number of sequences per sample and the percentage of sequences annotated at the species level were evaluated using a home-made R script.

MiSeq Illumina amplicon sequencing

The number of reads per sample and per sequencing (V3-V4 regions of the 16S rRNA gene and the ITS2 region of the ITS) is presented in the Table S1. Samples with less than 6000 reads were removed. Samples from the same season were pooled and rarefaction curves per season were done (Fig. S2).

Estimation of the trophic mode of the fungal species

We used the FUNGuild software [START_REF] Nguyen | An open annotation tool for parsing fungal community datasets by ecological guild[END_REF] to assign the trophic mode of the fungal species (RDP classifier based annotation). Fungal species annotated to a trophic mode with the level confidence "Possible" were grouped in the "not classified" fungi. Then, we calculated the percentage represented by each trophic mode per sample. Heatmaps were done using the R package gplots.

Chemical analyses

The elemental carbon (EC), organic carbon (OC), sugar anhydrides and alcohols [START_REF] Dommergue | Methods to investigate the global atmospheric microbiome[END_REF] .

(

Meteorological data

Meteorological parameters such as wind speed and direction, temperature, relative humidity and UV were collected (Vaisala instrument). For each sample, the backtrajectories of the air masses were calculated over 3 days before the sampling using HYSPLIT [START_REF] Draxler | An Overview of the HYSPLIT_4 Modelling System for Trajectories, Dispersion, and Deposition[END_REF] (maximum height above ground level: 1 km).

Graphical and Statistical analyses

Environmental variables. For chemical species, air concentrations in ng per cubic meter of air were used in the analyses. The chemical table was log10-transformed to approach a Gaussian distribution (verified on a Q-Q plot and tested using the Shapiro-Wilk test), and a hierarchical cluster analysis (average method) was done on the Euclidean distance matrix using the vegan and ade4 R packages. Meteorological data were used to do the wind roses using the openair R package [START_REF] Carslaw | Tools for the Analysis of Air Pollution Data[END_REF] . Backtrajectories of the air masses over three days were plotted on maps using the openair R package, and the relative surfaces of the landscapes (MODIS land surfaces) air masses over flown were calculated.

Diversity statistics and Multivariate analyses. Before doing the multivariate analyses, the raw abundances of the taxa (bacterial genera and fungal species) were transformed into relative abundances to counter the heterogeneity in the number of sequences per sample. A hierarchical cluster analysis (average metric) on the Bray-Curtis dissimilarity matrix was done using the vegan and ade4 R packages [START_REF] Dray | Analysis of Ecological Data: Exploratory and Euclidean Methodsin Environmental Science[END_REF] . We have defined the seasons as following: we

Results

Temporal distribution of airborne microbial communities

Airborne microbial abundance. Airborne bacterial and fungal concentrations (estimated by the number of 16S rRNA and 18S rRNA gene copies) were positively correlated (r=0.77, pvalue=7.9×10 -11 ) and varied between 1.8×10 3 and 2.1×10 7 cells per cubic meter of air and 3 and 1.0×10 5 cells per cubic meter of air, respectively. Neither bacteria nor fungal abundance significantly varied with season (pvalues=0.20 and 0.38, respectively), but the highest concentrations observed occurred in spring and summer for both bacteria and fungi (Table 1 and Fig. S4). and 897 bacterial genera and 97 and 820 fungal species. The estimated bacterial richness was higher in summer compared to winter and autumn (pvalue=4.5×10 -4 ; Table 1). The estimated fungal richness also was highest in summer, while not significant due to relatively higher variability (pvalue=0.18; Table 1). A rough correlation was found between the estimated richness estimations and concentration estimations for both bacteria and fungi (r=0.41, pvalue=3.0×10 -3 and r=0.50, pvalue=4.6×10 -4 , respectively).

Consistently with highest richness, summer had the highest percentage of unique bacterial genera (16% or 198/1239 of the genera in summer were only present in this season), followed by autumn (4.5% or 39/873), spring (4.4% or 42/953) and winter (4.3% or 37/864) (Fig. S5).

This was also observed for unique fungal species with the highest percentage observed in summer (28.4% or 457/1606), followed by autumn (17.4% or 191/1097), spring (14.4% or 172/1196) and winter (8% or 63/786) (Fig. S5). The majority of the bacterial genera and fungal species observed was present throughout the year, (i.e. they were shared by all the seasons; Fig. S5).

The five most abundant bacterial genera were Bacillus (15.6% in average) (Firmicutes), Hymenobacter (9.2%) (Bacteroidetes), Sphingomonas (7.4%) (Alphaproteobacteria), Methylobacterium (5.1%) (Alphaproteobacteria) and Thermoactinomyces (4.6%) (Firmicutes). Their relative abundance varied over the year: Bacillus was abundant throughout the year and dominated the community from summer to spring, while Hymenobacter increased in autumn and winter (19.2% and 13.9% respectively). The twenty-five most abundant bacterial genera and fungal species observed in each season are listed in Table S2.

In fungi, the five most abundant species were Pseudotaeniolina globosa (11.5% in average),

Cladophialophora proteae (7.9%), Alternaria sp. BMP_2012 (4%), Cladophialophora minutissima (4%) and Naevala minutissima (3.1%). Similar to bacteria, their relative abundance varied seasonally: Cladophialophora proteae represented 13.3% of the community in winter and only 0.3% in summer, when Alternaria dominated (10.8%); autumn was dominated by Pseudotaeniolina globosa and Cladophialophora proteae (16.4% and 15.6%, respectively), while Naevala minutissima was detected only in spring and summer, dominating in spring (10.1%).

Airborne microbial structure. The hierarchical cluster analyses of the samples based on both the bacterial (genus level) and fungal (species level) community structures are shown in Fig. 2; these were highly similar (r=0.50 and pvalue<0.001). The samples tended to group by season (anosim r=0.30 and r=0.49 with pvalues=0.001 for bacterial and for fungal communities, respectively; Fig. 2) with season explaining 17% and 22% of the variability in the community structures, respectively (pvalues=0.001). The distribution of the summer samples based on fungal species was different between 2016 and 2017 (pvalue=0.001). The intraseasonal variability in community structure was greatest in spring and smallest in summer for bacteria and conversely greatest in autumn and smallest in winter for fungi (Table S3). The relative abundances of fungal saprotrophs and symbiotrophs (like Balospora myosura, Strobilurus albipilatus, Pseudotaeniolina globosa) (Fig. 3) were greater in autumn and winter than during the rest of the year (pvalue=4.3×10 -5 and 0.5 for saprotrophs and symbiotrophs, respectively). The relative abundance of fungal pathotrophs was greater in the spring and summer periods (pvalue=0.51 -Fig. 3). Examples of phytopathogens increasing in relative abundance during the summer and/or spring are Mycosphaerella graminicola (wheat plant pathogen -Fig. S6), Microdochium majus (cereal pathogen -Fig. S6), Blumeria graminis (powdery mildew pathogen -Fig. S6), Ustilago hordei (maize pathogen -Fig. S6),

Botryotinia fuckeliana (gray mold disease) and Erysiphe alphitoides (powdery mildew on oak trees). However, the number of fungal species associated to pathogenic and saprophytic trophic modes was greatest in spring and summer, whereas symbiotrophs were mostly found in summer and autumn (Table S4). The season corresponding to each sample is indicated as a frieze (black=winter, blue=summer, green=spring and brown=autumn) that has been split into three based on the three major clusters observed on the hierarchical cluster analyses. When looking at the fifty most abundant bacterial genera and fungal species of the dataset that mainly controlled the distribution of the samples based on the microbial community structure (they represented 79.6% or 881898 sequences/1107323 and 71.8% or 367440 sequences/511738 of all the bacterial and fungal sequences, respectively), we observed three different patterns in the temporal variation of their relative abundance (Fig. 4 and5). The period during which the relative abundance increased varied in length and depended on the microorganism. First, some taxa were mostly abundant during the cold period of the year: 20% (10/50) of the bacterial genera and 34% (17/50) of the fungal species showed a higher relative abundance in winter and/or autumn compared to the other seasons. This was the case with Hymenobacter and Pseudotaeniolina proteae for example, whose relative abundances suddenly increased between November 2016 and February 2017, and between October 2016 and April 2017, respectively (Fig. 4a). Fungal taxa belonging to this group were almost exclusively saprotroph and/or pathotroph (Table S5). Second, some taxa were mostly abundant during summer and/or spring: 42% (21/50) of the bacterial genera and 46% (23/50) of the fungal species. For example, Sphingomonas and Alternaria sp. BMP_2012 relative abundances started to decrease in late summer/early autumn and increased again in the late spring (Fig. 4b). Fungal taxa belonging to this group were pathotroph, symbiotroph or saprotroph (Table S5). Finally, some taxa had no clear pattern in their relative abundance (i.e.

stable or randomly variable over the year): 38% (19/50) of the bacterial genera and 20%

(10/50) of the fungal species (e.g., Bacillus and Phaeosphaeria herpotrichoides) (Fig. 4c).

Fungal taxa belonging to this group were mostly saprotroph (Table S5). Temperature, wind speed and UV radiation (weekly averages) varied over the year (pvalue<0.05), conversely to wind direction and relative humidity. The mean weekly temperature varied between -5.5°C and 17.6°C (Fig. S3) and was significantly correlated to the season (pvalue=1.4×10 -11 -Fig. S10). Wind speed varied between 2.8 and 13.5 m/s on average, and this was significantly higher in winter compared to summer and spring (pvalue=2.1×10 -3 , Fig. S10), and significantly more variable in winter compared to the other seasons (pvalue=5.4×10 -4 ). Wind speed and direction were positively correlated (r=0.44, pvalue=0.001) with stronger winds coming from West. Nevertheless, the mean wind direction was not significantly different between seasons (pvalue=0.11) and the air mass origins were only partially correlated to the seasons (16% and 14% of the variance explained, pvalue=0.013). The wind roses and the backtrajectory density plots for each season are presented in Fig. S10 and Fig. S11. The seasonal weekly variability in wind conditions, temperature and relative humidity are shown in Table S3.

Bacterial concentration correlated best with UV radiation (r=0.38, pvalue=5.4x10 -3 ) and fungal concentration correlated best with temperature (r=0.38, pvalue=5.6×10 -3 ) (Table S8).

A significant and strong correlation was also found between both the bacterial and fungal community structures and the overall meteorology (i.e. temperature, relative humidity, wind direction and speed) (r=0.57 pvalue=0.001 and r=0.48 pvalue=0.001). Bacterial richness was strongly correlated with temperature (r=0.52, pvalue=7.7×10 -5 ) while fungal richness best correlated with wind direction (r=0.50, pvalue=4.6×10 -4 ) (Table S8).

Discussion

Seasonal variation in airborne microbial community composition due to changes in local landscapes

Previous investigations of the temporal variations of airborne microbial communities have consistently shown a seasonal shift in both cell concentration and community structure 2,9-12 .

This was explained mainly by changes in the surrounding landscape condition [START_REF] Bowers | Seasonal variability in airborne bacterial communities at a high-elevation site[END_REF][START_REF] Bowers | Seasonal variability in bacterial and fungal diversity of the near-surface atmosphere[END_REF][START_REF] Amato | An important oceanic source of micro-organisms for cloud water at the Puy de Dôme (France)[END_REF] (e.g., vegetation in summer, snow cover in winter etc.), local meteorology [START_REF] Uetake | Seasonal changes of airborne bacterial communities over Tokyo and influence of local meteorology[END_REF][START_REF] Innocente | Influence of seasonality, air mass origin and particulate matter chemical composition on airborne bacterial community structure in the Po Valley, Italy[END_REF] and/or the origin of the air masses (i.e. changes in the global air circulation) [START_REF] Innocente | Influence of seasonality, air mass origin and particulate matter chemical composition on airborne bacterial community structure in the Po Valley, Italy[END_REF] . Our data showed that seasonality was correlated to the temporal distribution of airborne microbial community structure at puy de Dôme. With these results, we demonstrated the importance of local regional sources (i.e.

within 50 km) even at relatively elevated sites such as puy de Dôme. Puy de Dôme is mainly surrounded by croplands and/or natural vegetation (> 80% of the surface within a 50 km perimeter based on MODIS satellite images; Fig. 1) that undergo strong changes over the year at such latitudes (see puy de Dôme monthly satellite images in Fig. S1 for an overview).

These changes are partly associated with crops (mainly wheat and grain maize) and vegetation that cycle through their different phases on an annual basis. Changes in landscape characteristics are themselves under direct influence of climatic conditions, including precipitation rate, temperature and sunlight exposure. This consequently has a critical influence on the surface microbial communities [START_REF] Zhang | Alterations in soil microbial community composition and biomass following agricultural land use change[END_REF][START_REF] Constancias | Mapping and determinism of soil microbial community distribution across an agricultural landscape[END_REF] . Over the year, different microorganisms with different trophic modes were likely aerosolized (Fig. 3). Fungal phytopathogens (like Ustilago hordei) and leaf-associated fungi (like Naevala minutissima) increased in relative abundance during the spring and summer periods when the crop plants grew and trees were green; while saprotrophs (like Cladophialora protea) increased in relative abundance during the autumn and winter periods after crop harvesting and when dead and decomposing biological material covered terrestrial surfaces. Similar to fungi, soil-associated bacterial taxa (like Hymenobacter) were observed in higher relative abundance in autumn and winter and bacterial phytopathogens and leaf-associated bacteria (like Sphingomonas) were observed in higher relative abundance in spring and summer. The three general trends (i.e. an increase of specific taxa in autumn/winter; an increase of other taxa in summer/spring; stable or randomly variable microbial taxa over the year -Fig. 4) might have driven the distribution of the samples based on both the airborne bacterial and fungal community structures in two major clusters (i.e. an autumn/winter cluster versus a spring/summer cluster observed in Fig. 2). In addition, the period during which we observed an increase in the relative abundance of a winter or summer-associated microbial taxon varied in length and might have been specific to the life strategy of the given taxon. Microbial taxa whose relative abundance remained stable or varied randomly over the year might partly belong to taxa associated to decomposing matter (the trophic mode of all the fungal taxa present in this group was saprotroph) that is present the whole year. Airborne fungal community structure showed a stronger seasonal variation than bacterial community structure did, likely related to a higher number of fungal taxa showing a seasonal pattern as compared to bacterial taxa (40/50 and 31/50 for fungi and bacteria, respectively). The stronger seasonal shift observed in airborne fungi might be explained by a greater influence of vegetation on fungi as compared to bacteria [START_REF] Sun | Soil Bacterial and Fungal Communities Show Distinct Recovery Patterns during Forest Ecosystem Restoration[END_REF] .

Role of local meteorology in the intraseasonal variability of airborne microbial community composition

Local meteorology might also play a role in the temporal distribution of airborne microbial communities at puy de Dôme especially on their intraseasonal variability. In a previous study considering numerous sampling sites 1 , we highlighted the importance of local meteorology (especially wind speed, wind direction and temperature variability over time) on the variability of airborne microbial communities when the site was surrounded by a variety of landscapes. Puy de Dôme is characterized by strong wind speeds and highly variable meteorological conditions partly explained by its central position and its high elevation within the Chaîne des Puys mountain range (+1465 m altitude above sea level and up to 500 m above surrounding landscapes). The wind direction changed rapidly both within and between the weeks. The relative humidity and temperature also showed a high variability within and between the weeks within the same season (Table S3). While the correlation between meteorological parameters and airborne microbial communities at puy de Dôme is mainly explained by seasonality, local meteorological parameters might have also affected airborne microbial structures by influencing the aerosolization process as discussed in Tignat-Perrier et al. (2019) [START_REF] Tignat-Perrier | Global airborne microbial communities controlled by surrounding landscapes and wind conditions[END_REF] . For example, specific temperatures might activate the sporulation of specific fungi [START_REF] Pickersgill | Lifestyle dependent occurrence of airborne fungi[END_REF] . The high correlation observed between temperature and airborne bacterial richness might be explained by the increased turbulence in air, a higher microbial diversity in the sources (i.e. surrounding landscapes) [START_REF] Dennis | Soil bacterial diversity is positively associated with air temperature in the maritime Antarctic[END_REF][START_REF] Zhou | Temperature mediates continental-scale diversity of microbes in forest soils[END_REF][START_REF] Nottingham | Microbes follow Humboldt: temperature drives plant and soil microbial diversity patterns from the Amazon to the Andes[END_REF][START_REF] Tittensor | Global patterns and predictors of marine biodiversity across taxa[END_REF] as well as a drier surface favoring cell and biofilm detachment. Wind parameters were the meteorological parameters that best correlated to airborne fungal richness. Stronger winds are obviously more likely to aerosolize particles from surfaces.

Minor contribution of distant sources in the temporal variation of airborne microbial communities

Weekly averages of wind direction and origin of the air masses did not drastically change throughout the seasons and were highly and randomly variable over the year (Fig. S10 and S11). We did not observe correlations between airborne microbial community structure and the origin of the air masses. In winter, during which free troposphere air masses, and thus potentially long-range transport, have a larger influence [START_REF] Venzac | Seasonal variation of aerosol size distributions in the free troposphere and residual layer at the puy de Dôme station, France[END_REF][START_REF] Bourcier | Seasonal variation of water-soluble inorganic components in aerosol size-segregated at the puy de Dôme station (1,465 m a.s.l.), France[END_REF][START_REF] Freney | Seasonal variations in aerosol particle composition at the puy-de-Dôme research station in France[END_REF] , the variability in the bacterial community structure was not the highest. Marine air masses have been shown to prevail (72%) during winter at puy de Dôme [START_REF] Freney | Seasonal variations in aerosol particle composition at the puy-de-Dôme research station in France[END_REF] . One sample showed an especially strong marine signature in terms of chemistry (February 2 nd , 2017). However, this sample has similar proportion of marine bacterial genera (Corialomargarita, Rubritalea, Aquimarina) as other samples. Whenever present, the identified specific marine bacterial genera were very rare (they were represented by only 2 or 3 sequences over > 20000 sequences per sample). Unlike previous suggestions for cloud water-associated microorganisms at this site, microbial communities in the dry phase did not seem to mainly originate from oceanic sources [START_REF] Amato | An important oceanic source of micro-organisms for cloud water at the Puy de Dôme (France)[END_REF] We also observed a seasonal change in the particulate matter chemistry (PM10 chemistry) (Fig. S7). At puy de Dôme, changes in PM10 chemistry throughout the seasons were correlated to changes in air mass origins (local and distant) and changes in the vertical stratification of the atmospheric layers (such as the increase of the height of the mixed layer in summer) [START_REF] Venzac | Seasonal variation of aerosol size distributions in the free troposphere and residual layer at the puy de Dôme station, France[END_REF][START_REF] Bourcier | Seasonal variation of water-soluble inorganic components in aerosol size-segregated at the puy de Dôme station (1,465 m a.s.l.), France[END_REF] . We observed specific and strong correlations between some chemical species and microbial taxa (Table S7) and this could explain the overall correlation between the overall PM10 chemistry and airborne microbial structure. In particular, we observed correlations between specific microbial taxa and polyols. The concentration of atmospheric polyols might be due to the presence of airborne green plant debris as well as specific microbial taxa and especially fungal taxa which produce polyols [START_REF] Medeiros | Sugars as source indicators of biogenic organic carbon in aerosols collected above the Howland Experimental Forest, Maine[END_REF][START_REF] Ruijter | Mannitol Is Required for Stress Tolerance in Aspergillus niger Conidiospores[END_REF][START_REF] Solomon | Decoding the mannitol enigma in filamentous fungi[END_REF][START_REF] Lewis | Sugar alcohols (polyols) in fungi and green plants[END_REF] . Our sampling strategy (one-week sampling) might not have been adapted to identify microbial long-range transport events that, if they occurred, might have been greatly diluted by the local sources.

Investigations on the temporal variability of airborne microbial communities that concluded the significant contribution of distant sources in the composition of airborne microbial communities [START_REF] Innocente | Influence of seasonality, air mass origin and particulate matter chemical composition on airborne bacterial community structure in the Po Valley, Italy[END_REF][START_REF] Cáliz | A long-term survey unveils strong seasonal patterns in the airborne microbiome coupled to general and regional atmospheric circulations[END_REF] had either a different sampling strategy than ours (i.e. shorter sampling duration such as 24 h 9 ) or did not sample microorganisms of the dry phase of the troposphere [START_REF] Cáliz | A long-term survey unveils strong seasonal patterns in the airborne microbiome coupled to general and regional atmospheric circulations[END_REF] .

Conclusion

We investigated changes in airborne microbial community concentration and structure at the elevated continental site puy de Dôme in the Massif Central of France (+1465 m altitude above sea level) as a function of the local meteorology and particulate matter chemistry. We showed that puy de Dôme airborne microbial community structure likely shifted throughout the year in relation to the seasonal changes in surface conditions of the surrounding landscape of the mountain that was characterized mainly by croplands and natural vegetation. The microbial taxa that drove the seasonal distribution of airborne microbial communities showed different trends throughout the seasons depending on their trophic mode. Crop-associated microorganisms and especially crop pathogens were in higher relative abundance in spring/summer while soil-associated microorganisms and dead material-associated microorganisms were found in higher relative abundance in autumn/winter. Changes in the vertical stratification of the atmospheric layers throughout the seasons (such as the increase of the height of the mixed layer in summer) might also play a role in the seasonal shift of puy de Dôme airborne microbial communities. Within the different seasons, the temporal variability observed in the composition of airborne microbial communities was likely associated to the variable and windy meteorological conditions observed in puy de Dôme.

Fig. 1 .

 1 Fig. 1. Geographical location and landscape of the sampling site. Map showing the

  adjusted the beginning and ending administrative dates of the seasons (i.e. summer: 20 th of June to 22 th of September; autumn: 23 th of September to 21 th of December; winter: 22 th of December to 19 th of March; spring: 20 th of March to 19 th of June) based on the weekly mean temperature (Fig.S3). France goes through a cycle of four strong seasons characterized by distinctive weather: the summer is characterized by higher temperatures, a longer daylight period and the period of fructification of fruiting plants; the autumn is the harvest period of most annual crops and a preparation period for dormancy; winter is characterized by the lowest temperatures, frequent precipitation (rain and snow) and high air humidity; and finally the spring is characterized by milder weather, snow melting, budding and flowering. To access the variability of microbial population structure within the seasons, we averaged the degrees of dissimilarity obtained from the Bray-Curtis matrix for each pair of samples from the same season, subtracted these values from 1 to get similarity values and divided the similarity values by the standard deviation (as detailed in Tignat-Perrier et al., 2019 1 ). Spearman correlations were calculated to test the correlation between microbial abundance and richness and quantitative environmental factors using the Hmisc R package[START_REF] Harrell | Harrell Miscellaneous -Package 'Hmisc[END_REF] . ANOVA analyses were used to test the influence of qualitative factors such as season and year on both bacterial and fungal abundance and richness using the vegan R package, followed by TukeyHSD tests to identify which group revealed a significantly different mean. A distancebased redundancy analysis (RDAlinear or non-linear correlation) was carried out to evaluate the part of the variance between the samples explained by the seasons, chemistry and/or meteorology, and an ANOVA was carried out to test each variable using the vegan and ade4 R packages. Venn diagrams using the R package VennDiagram were done to access the shared and unique bacterial genera and fungal species from each season after rarefaction on the raw abundances (rarefaction at the minimum number of reads). A Mantel test between the Bray-Curtis matrices based on the bacterial genera and fungal species was used to evaluate similarities in the distribution of the samples. A Mantel test was done between the Bray-Curtis matrix based on either the bacterial or the fungal diversity and the Euclidean distance matrix based on the chemical variables or meteorology to evaluate the similarities in the distribution of the samples.

Fig. 2 .

 2 Fig. 2. Distribution of the samples based on the microbial community structure.

Fig. 3 .

 3 Fig. 3. Distribution of the trophic modes of the fungal species identified. Each fungal

Fig. 4 .

 4 Fig. 4. Temporal evolution of the relative abundance of several fungal species and

Fig. 5 .

 5 Fig. 5. Temporal evolution of the relative abundances of the dominant microbial taxa.

  . Uetake et al. (2019) 2 investigated airborne (not cloud-associated) microbial communities in Tokyo (Japan) over a year and also highlighted the absence of oceanic related bacteria (i.e. SAR group, Oceanospirillales) that would have suggested transport of airborne microbial cells from the Pacific Ocean to Tokyo.

16S rRNA gene sequencing: library preparation, reads quality filtering and taxonomic annotation. The

		V3-V4 region of the 16S rRNA gene was amplified and libraries were
	prepared as presented in Tignat-Perrier et al. (2019) 1 . The amplicons were sequenced by
	paired-end MiSeq sequencing using the V3 Illumina technology with 2 x 250 cycles. Reads
	were	filtered	based	on	quality	using	FASTX-Toolkit
	(http://hannonlab.cshl.edu/fastx_toolkit/), assembled using PANDAseq 19 , and annotated using
	RDP Classifier 20 and the RDP 16srRNA database as detailed in Tignat-Perrier et al. (2019) 1 .
	RDP classifier was used in part to avoid errors due to sequence clustering. The number of
	sequences per sample and the percentage of sequences annotated at the genus level were
	evaluated using a home-made R script. The sequences annotated as chloroplasts by RDP were
	manually removed.

ITS rRNA gene sequencing: library preparation, reads quality filtering and taxonomic annotation.

	The ITS2 region of the ITS was amplified libraries were prepared as presented in
	Tignat-Perrier et al. (2019) 1 . The amplicons were sequenced by a paired-end MiSeq
	sequencing using the technology V2 of Illumina with 2 x 250 cycles. Reads were filtered
	based on quality using FASTX-Toolkit, assembled using PANDAseq, and annotated at the
	species level 21 using RDP Classifier and the RDP fungallsu database as detailed in Tignat-
	Perrier et al. (2019)

Table 1 . Microbial concentration and richness estimations depending on the season.

 1 Concentrations of bacterial and fungal ribosomal gene copies and associated richness (number of bacterial genera and fungal species) averaged for each season and relative standard deviation. Reference letters indicate the group membership based on Tukey's HSD post hoc tests (done only for the estimated bacterial richness as the ANOVA showed differences between the groups).

	Season	Average bacterial concentration (number of 16S rRNA gene copies/m 3 of air)	Average fungal concentration (number of 18S rRNA gene copies/m 3 of air)	Average Chao1 bacterial richness estimation (estimated number of bacterial genera)	Average Chao1 fungal richness estimation (estimated number of fungal species)
	PDD spring 2.7×10	6 +/-6.2×10	6	4.6×10	3 +/-1.1×10	4	4.8×10	2 +/-1.4×10	2 ab	4.7×10	2 +/-9.3×10	1
	PDD summer	8.5×10	5 +/-1.3×10	6	1.6×10	4 +/-2.8×10	4	6.1×10	2 +/-1.5×10	2 a	4.6×10	2 +/-1.9×10	2
	PDD autumn	2.2×10	5 +/-4.0×10	5	3.2×10	3 +/-4.6×10	3	4.3×10	2 +/-1.2×10	2 b	3.6×10	2 +/-1.9×10	2
	PDD winter 3.6×10	5 +/-9.0×10	5	7.4×10	2 +/-9.4×10	2	3.9×10	2 +/-1.4×10	2 b	3.0×10	2 +/-1.8×10	2
	Airborne microbial richness. The observed richness varied between 150 and 674 genera of
	bacteria, and 84 and 649 species of fungi. The estimated richness (Chao1) varied between 234

Acknowledgements

This program was funded by ANR-15-CE01-0002-INHALE, Région Auvergne-Rhône Alpes and CAMPUS France. The chemical analyses were performed at the IGE AirOSol platform.

We thank L.Pouilloux for computing assistance and maintenance of the Newton supercalculator.

Data availability

Sequences reported in this paper have been deposited in ftp://ftp-adn.eclyon.fr/aerobiology_Puy-de-Dome_amplicon_INHALE/. A file has been attached explaining the correspondence between file names and samples.

Atmospheric PM10 chemistry

The hierarchical cluster analysis of the samples based on the PM10 chemical profile is shown in Fig. S7. Chemical concentrations varied significantly with season (pvalues<0.05) for all the compounds measured except Cl -, NO 3 -, NH 4 + and rhamnose (pvalues>0.05) (Table S6).

Seasons explained 38% of the variance in the distribution of the samples based on the chemical composition (pvalue=0.001). When looking at the weekly variation of the concentrations of the different compounds, we observed general trends of seasonal variations (as observed for microbial communities). Some increased during autumn/winter compared to spring/summer (galactosan, levoglucosan and mannosan), while some others decreased (erythriol, glucose, trehalose, sorbitol, xylitol, inositol, SO

, OC, MSA) (Fig. S8). The sample distribution based on bacterial and fungal community structures were correlated to their distribution based on the overall PM10 chemistry (Mantel test r=0.25 pvalue=0.001 and 0.41 pvalue=0.001, respectively) (see RDA analyses in Fig. S9). Specific chemical compounds were the main contributors to these correlations (i.e. the polyols, MSA and Na + for the fungal communities, and organic carbon, Na + , Cl -and erythriol for the bacterial communities). Consistent with seasonal patterns observed for both microbial community structures and chemical composition, we observed strong positive and negative correlations between the concentration of some chemical compounds and the relative abundances of bacterial genera or fungal species. For examples, mannitol-arabitol and Alternaria sp. BMP_2012 (r=0.73 and pvalue=4.8×10 -9 ) and mannitol-arabitol and Pseudotaeniolina globosa (r=-0.66 and pvalue=5.6×10 -7 ) (Table S7).

Weather characteristics: local meteorology and air mass geographical origin

Competing interests

The authors declare no competing interests.

Author contribution AD, PA, CL and TMV designed the experiment. PA, AT, MJ and KS conducted the sampling field campaign. RTP did the molecular biology, bioinformatics and statistical analyses. RTP, AD, CL, TMV analyzed the results. RTP, TM, AD, PA and CL wrote the manuscript. All authors reviewed the manuscript.


Mélanie Guyot

Thomas Simon

Franck Ceppo

Clara Panzolini

Alice Guyon

Julien Lavergne

Emilie Murris

Douglas Daoudlarian

Romain Brusini

Hadi Zarif

Sophie Abelanet

Sandrine Hugues-Ascery

Jean-Louis Divoux

Stephen J Lewis

Arun Sridhar

Nicolas Glaichenhaus

Philippe Blancou

Pancreatic nerve electrostimulation inhibits recent-onset autoimmune diabetes

Keywords: electrostimulation, autoimmune diabetes, T-cells, cross-presentation, leukocyte migration, inflammation

Electrical modulation of peripheral nerves has been proposed as a novel approach to treat immune-mediated inflammatory diseases (IMIDs) [START_REF] Birmingham | Bioelectronic medicines: a research roadmap[END_REF] . Type-1 diabetes (T1D) is an autoimmune disease that results from the destruction of insulin-producing pancreatic β cells by autoreactive T lymphocytes following their activation in pancreatic draining lymph nodes (LNs) [START_REF] Herold | Restoring immune balance in type 1 diabetes[END_REF] . The sympathetic nervous system inhibits inflammation via binding of noradrenaline or adrenaline to β 2 -adrenergic receptors (ARs) expressed on immune cells [START_REF] Padro | Neuroendocrine regulation of inflammation[END_REF] . Since LNs are innervated by sympathetic nerves, we hypothesized that pancreatic nerve electrical stimulation (PNES) may inhibit local inflammation and T1D progression.

Here we have developed a minimally invasive surgical procedure for long-term implantation of micro-cuffs electrodes onto the nerve that projects to pancreatic draining LNs. When applied to recently diabetic Non-Obese-Diabetic (NOD) mice, PNES inhibited insulitis and T1D progression with minimal bystander tissue effects by inhibiting the migration of pathogenic effector T lymphocytes from pancreatic draining LNs to pancreatic islets. Our data provide a rationale for the use of bio-electronic medicine as a potential treatment modality for T1D.

Little is known about the nerves that project to the pancreas in mice. Gross anatomy of the pancreatic region showed that the superior mesenteric artery branching from the inferior vena cava is associated with a 50 µm-diameter nerve-like structure (Suppl. Fig. 1a) and supplied blood to the pancreas head and neck (Suppl. Fig. 1b). To confirm that this structure was a nerve and to identify the innervated tissues, we placed a hook electrode onto it and a recording electrode on visceral tissues. Evoked compound action potentials (CAPs) occurred in pancreatic LNs and pancreas head but not in liver when electrostimulation was applied (Suppl. Fig. 2). Pancreatic nerve-like structures expressed tyrosine hydroxylase (TH) demonstrating the presence of catecholaminergic fibers (Fig. 1a). In agreement with the known ability of the SNS to induce vasoconstriction, high frequency and amplitude (20 Hz, 1 mA) electrical stimulation of the pancreatic nerve reduced pancreatic blood flow using laser speckle perfusion imager (Fig. 1b). Light-sheet-based fluorescent imaging of TH-stained LNs showed a high-density network of catecholaminergic fibers showing that the pancreatic nerve did not only project to the pancreas itself, but also to the LNs that drained this gland (Fig. 1c). To characterize the electrophysiological properties of the pancreatic nerve fibers, we used fluorescent reporter tdTomato TH-Cre transgenic mice carrying the tdTomato fluorescent protein gene downstream of the tyrosine hydroxylase (TH) gene promoter. Red fluorescence axons were readily detected in both the pancreatic nerve (Suppl. Fig. 3a) and the pancreatic LN medulla zone (Suppl. Fig. 3b). To confirm that the pancreatic nerve projected to the pancreatic LN, we placed these red fluorescent axons of the pancreatic nerve into a suction electrode and recorded field action potentials (FAPs) with a microelectrode. FAPs were readily detected when pancreatic nerve electrical stimulation (PNES) was above 400 µA (Fig. 1d). FAPs were inhibited by tetrodotoxin (TTX) confirming that they resulted from neuronal activity (Suppl. Fig. 4a). Nerve conduction velocity was 0.5 ± 0.26 m/s at 25 °C (n = 4) suggesting that axons were unmyelinated [START_REF] Duclaux | Conduction velocity along the afferent vagal dendrites: a new type of fibre[END_REF] .

Should PNES be considered as a therapeutic treatment, electrical parameters need be optimized to minimize bystander tissue damage and short-term alterations in pancreatic blood flow and glycemia, while allowing release of therapeutic levels of neurotransmitters. After testing various electrical stimulation parameters, we found that a frequency of 10 Hz and an amplitude of 450 µA increased noradrenaline levels in pancreatic LNs (Fig. 1e) without altering pancreatic blood flow (Fig. 1b) or glycemia (Suppl. Fig. 5). Furthermore, these parameters did not induce exhaustion of axonal excitability (Suppl. Fig. 4b,4c) therefore preserving therapeutic efficacy.

We then investigated whether PNES could impact secretion of pro-inflammatory cytokines, immune cell trafficking and antigen cross-priming in the pancreatic draining LNs of non-diabetic prone mice. Because anesthetics interfere with immunity [START_REF] Fuentes | General anesthesia delays the inflammatory response and increases survival for mice with endotoxic shock[END_REF] , we developed a minimally invasive surgical procedure for sustainable implantation of micro-cuffs electrodes onto the pancreatic nerve allowing studies to be conducted on conscious animals. Compared to sham-stimulated mice in which a cuff was implanted but electrical stimulation was not applied, PNES mice exhibited higher numbers of T and B-lymphocytes in pancreatic draining but not in non-draining LNs (Fig. 2a, Suppl. Fig. 6a-c). No alterations in the frequency of myeloid cell types were observed Suppl. Fig. 6d). One session of PNES reduced LPS-induced proinflammatory cytokine mRNA levels in pancreatic LNs but not in spleen (Fig. 2b). We next assessed the impact of electrostimulation on the ability of dendritic cells (DC) to cross-prime CD8+ T cells taking advantage of the RIP-OVA mice in which ovalbumin (OVA) is exclusively expressed in β-islets. In this model, naïve OVA-specific CD8 T (OT-1) cells transferred in these mice spontaneously proliferate as consequence of OVA antigen presentation by DC in pancreatic lymph nodes. When applied over a 4-day period, PNES inhibited pancreatic autoantigen cross-priming as shown by reduced proliferation of adoptively transferred islet-specific CD8 + T cells (Fig. 2c). In agreement with a critical role of noradrenaline, the effects of PNES on both immune cell accumulation and LPS-induced pro-inflammatory cytokine mRNA production were abolished in mice deficient in β 2 -AR (ADRB2 ko ) (Fig. 2a,2b). Likewise, administration of the β 1 / β 2 -AR antagonist propranolol showed that the inhibitory effect of PNES on auto-antigen cross-priming was β-AR-dependent (Fig. 2c).

We next investigated the impact of PNES on T1D progression in NOD mice that spontaneously develop autoimmune diabetes between 3 and 6 months of age. Once diagnosed with diabetes, NOD mice were implanted with a micro-cuff electrode onto the pancreatic nerve and PNES was applied one day later three times a day for 6 weeks (Fig. 3a). Mice returned to normoglycemia after surgery, a phenomenon that was likely due to the anti-inflammatory effect of anesthetics [START_REF] Lee | Isoflurane improves survival and protects against renal and hepatic injury in murine septic peritonitis[END_REF][START_REF] Lee | Isoflurane protects against renal ischemia and reperfusion injury and modulates leukocyte infiltration in mice[END_REF] . However, while glycemia started to increase again in sham stimulated mice resulting in full-blown diabetes, it remained below 150 mg/ml in PNES mice (Fig. 3b). To investigate the underlying mechanisms, we took advantage of a well-characterized synchronous model of diabetes in which diabetogenic T cells from NOD mice are transferred into immunodeficient syngenic NOD-SCID recipients [START_REF] Christianson | Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors[END_REF] . While all (13 out of 13) sham-stimulated mice developed T1D within 4 weeks after transfer, only 55% (7 out of 13) of PNES recipients did (p = 0.015, Fig. 3c). This result showed that PNES inhibits T1D progression by acting on pathogenic effector T cells. In another set of experiments, mice were sacrificed 2 weeks after adoptive transfer and analyzed for both insulitis and number of lymphocytes in pancreatic LNs.

PNES mice had 2.7-fold as many islets than sham stimulated mice (84.5 ± 20.3 versus 31.75 ± 3.6, p = 0,029) (Fig. 3d). In addition, the proportion of non-infiltrated islets was higher in PNES mice than in sham stimulated mice (55% versus 17.5%)(Fig. 3d). Furthermore, while PNES and sham-stimulated mice exhibited similar number of lymphocytes in non-draining LNs, PNES mice had 4-to 5-fold more lymphocytes in draining LNs (29.0 ± 12.0 x 10 4 versus 6.0 ± 1.5 x 10 4 per mouse, p = 0.006), a result similar to what we also observed in C57BL/6 mice (Fig. 3e). Our data are compatible with a model in which PNES prevents T1D by inhibiting the migration of pathogenic effector T lymphocytes from pancreatic draining LNs to pancreatic islets.

We next investigated whether T1D inhibition could be achieved by on-demand PNES, or alternatively required persistent stimulation. Once diagnosed with diabetes, NOD mice were implanted with a micro-cuff electrode onto the pancreatic nerve and glycaemia was monitored daily for up to two months. When glycaemia exceeded 200 mg/dl, 9 to 12 PNES sessions (450 µA, 10 Hz, 2 min. duration) were applied 8 hours apart. While all mice eventually became hyperglycemic, glycaemia increased 2.25 times more slowly in PNES mice (Fig. 3f, Suppl. Fig. 7). In another set of experiments, we sacrificed mice 2 weeks after the first PNES session.

PNES mice had 4.3-fold as many islets as sham stimulated mice (26.0 ± 1.5 versus 6.0 ± 1.0 per mouse, p = 0.0006) (Fig. 3g). Likewise, insulitis was less severe in PNES mice as demonstrated by the proportion of non-infiltrated islets (30.9% versus 15.8%).

Two clinical studies have shown that vagus nerve stimulation can be beneficial for patients with Immune-Mediated Inflammatory Diseases (IMIDs), i.e. rheumatoid arthritis [START_REF] Koopman | Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis[END_REF] and Crohn's Disease [START_REF] Bonaz | Chronic vagus nerve stimulation in Crohn's disease: a 6-month follow-up pilot study[END_REF] . However, vagus nerve electrical stimulation lacked efficacy in some patients, and had serious adverse effects, presumably because of its lower nerve fiber specificity. Indeed, the vagus nerve contains both afferent and efferent fibers, and it regulates the function of multiple organs. We have made the hypothesis that applying electrical stimulation to an exclusively efferent nerve proximal to the target organ would be both more efficient therapeutically and more specific. Here, we have identified a sympathetic nerve that projects into pancreatic LNs. Selective electrostimulation of this nerve was sufficient to completely inhibit T1D progression in both a spontaneous and an adoptive transfer models of T1D with minimal bystander effects. We also found that PNES inhibited LPS-induced production of inflammatory cytokines, antigen cross-presentation in pancreatic LN, and resulted in increased numbers of lymphocytes in pancreatic draining LN. These results are in agreement with previous studies showing an inhibitory role of β2-AR signaling on secretion of inflammatory cytokines by various myeloid cell types 3 , antigen cross-presentation by dendritic cells [START_REF] Hervé | β2-Adrenoreceptor agonist inhibits antigen cross-presentation by dendritic cells[END_REF] and lymphocyte egress from LN [START_REF] Nakai | Control of lymphocyte egress from lymph nodes through β2-adrenergic receptors[END_REF] . Since T1D is dependent on antigen crosspresentation, production of inflammatory cytokines and migration of autoreactive effector T cells from pancreatic LNs to pancreatic islets 2 , PNES could possibly prevent T1D progression by interfering with all such pathways. Bioelectronics medicine offers the possibility to apply on-demand treatment based on recording of physiological parameters such as glycemia. While our pilot experiments showed that on-demand PNES was not as efficient as continuous PNES at inhibiting T1D progression, technological refinements such as optimized electrical stimulation parameters or implementation of a closed-loop system are within reach and may greatly improve its therapeutic efficacy. Thus, our study provides a rationale for the future use of bioelectronic medicine in treatment of autoimmune diabetes.

Figure 1 :

 1 Figure 1: Pancreatic nerve electrophysiological and functional characterization. (a)

Figure 2 :

 2 Figure 2: Impact of PNES on immune cell number, cytokine production and autoreactive

Figure 3 :

 3 Figure 3: Impact of PNES on glycaemia and insulitis in NOD mice. (a) Schematic

  profilerating cell (normalized to sham)

  CD8 OVA-specific cells i.v.

  figure f-h

Acknowledgements. We would like to thank Samah Rekima for technical assistance. This work was funded by a collaborative research grant from GSK Bioelectronics R&D. This work was also supported by the LABEX SIGNALIFE (#ANR-11-LABX-0028-01) and the FHU Oncoage.

Competing financial interest. A patent is pending on pancreatic nerve electrostimulation

Methods

Mice:

All experiments were performed with female C57BL/6, NOD, NOD-SCID mice (Charles River), or RIP-mOVA [START_REF] Kurts | Major histocompatibility complex class I-restricted cross-presentation is biased towards high dose antigens and those released during cellular destruction[END_REF] , OT-I 14 , ADRB2 ko [START_REF] Chruscinski | Targeted disruption of the beta2 adrenergic receptor gene[END_REF] and tdTomato TH-Cre 16, 17 mice backcrossed onto the C57BL/6 background for at least 10 generations. Mice were housed on a 12 hours light/dark cycle (lights on/off at 7 am/7 pm) with food ad libitum. Mice were treated in accordance with our local Animal Care and Use Committee guidelines.

Electrodes and surgery:

For studies in anaesthetized animals, mice were anaesthetized by an intraperitoneal injection of a mixture of ketamine (75 mg/kg) and xylazine (60 mg/kg) and a hook electrode was placed under the pancreatic nerve. For studies in conscious animals, mice were anaesthetized with isoflurane and the area around the right abdominal artery next to the kidney was exposed (Suppl Fig. 1a). One mm length 100 µm-sling micro-cuff electrodes (CorTec) were implanted onto the pancreatic nerve.

Recordings of CAP and FAP:

After placing a hook electrode onto the pancreatic nerve and artery, platinum-iridium recording electrodes (Phymep) were placed onto the pancreatic nerve, pancreatic lymph node, pancreas tissue and liver for CAP recording using a wireless recording system (W8, Multi-Channel Systems). Ground/Reference wires were placed into the nearby tissue. For FAP, recordings were performed on explants from tdTomato TH-Cre that were placed in a recording chamber at room temperature (20-25°C) superfused with oxygenated artificial cerebrospinal fluid (ACSF) under a microscope (Zeiss). The pancreatic nerve was introduced into a suction-stimulating electrode stimulated with square pulses of 1 ms and intensities of 10-1500 µA. Recordings were made using pipettes made from borosilicate glass capillary (Hilgenberg) with resistance of 3-6 MW when filled with extracellular solution, placed near red fluorescent axons. Signals were amplified using an Axopatch 200B (Axon Instruments), digitized at 10kHz via an Digidata 3200 interface (Molecular devices) controlled by pClamp10.0 software (Molecular Devices) and digitally filtered at 3KHz. CAP and FAP recordings were performed in a Faraday cage.

Electrostimulation: For studies on anaesthetized mice, Master-8 (A.M.P.I.), PlexStim V2.3 (Plexon) and STG 4002 stimulator (Multichannel system) were used respectively for CAP, pancreatic blood perfusion and FAP recording. For all studies in conscious animals, mice were placed in individual cage and connected to a PlexStim V2.3 (Plexon) or MAPS (Axonic) stimulator. Unless specified, the set-up of the electrostimulation were rectangular chargedbalanced biphasic pulses with 450 µA pulse amplitude, 2 ms pulse width (positive and negative) at 10 Hz frequency for 2 minutes. In propranolol-treated groups, mice were injected intraperitoneally with a 5mg/kg dose, 30 min before electrostimulation.

Noradrenaline levels: Pancreatic LNs were harvested and snap-frozen in liquid nitrogen immediately after electostimulation. The organ was processed and noradernaline was measured by ELISA (DLD Diagnostika GmbH) via manufacturer recommandations.

Flow Cytometry: Single-cell suspensions were stained with anti-CD45 (clone 30F11), anti-CD3 (17A2), anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-CD19 (1D3). All antibodies are from BD Biosciences. Dead cells were excluded using 7-AAD staining. Data were acquired on SP6800 (Sony) flow cytometer and analyzed using Kaluza software.

RT-PCR:

RNAs from pancreas were isolated via the manufacturer's instructions (miRNEasy micro kit, Quiagen), reverse transcribed using QuantiTect Reverse transcription kit (Quiagen) and amplicons were quantified SyberGreen Master Kit (Roche) and LightCycler 480 II (Roche). mRNAs cytokine expression were normalized to GAPDH using LightCycler software (Roche).

In vivo cross-priming assay:

Cross-priming in vivo was assessed in Rip-mOVA mice. 10 7 of CFSE-labeled OT-I CD8+ T cells were injected intravenously to these mice at day 0. Between day 1 and day 4, animals were electrostimulated or not (450 µA, 10 Hz, 2 minutes) 3 times a day (3 hours apart). On day 4, mice were sacrificed, pancreatic lymph node removed and CD8 + proliferation was assessed by flow cytometry using SP6800 (Sony). Rip-mOVA mice were treated with propranolol (5mg/kg ip) 30mn before each stimulation.

Adoptive cell transfers in NOD-SCID: Recipients were adult 6-week-old NOD-SCID mice.

Animals were injected intravenously with 5 x 10 6 splenocytes from overtly diabetic NOD mice.

Glycaemia follow-up: Glycaemia was monitored using a Free Style Papillon Vision (Abbott) taking a blood drop (<10 µl) from the tail. NOD and NOD-SCID mice were considered diabetic when glycaemia were over 250mg/dl for two successive days. To determine the increase slope of NOD mice linear regression to determine glycaemia including the first glycaemia time point over 200mg/dl and the 2 glycaemia time point exceeding 499mg/dl. Laser speckle imaging: After anaesthesia, the pancreas tissue was exposed and placed about 30 cm below the Moor-FLPI laser speckle perfusion imager (Moor instruments Ltd.). Then the pancreatic blood perfusion images were saved and analyzed by the Image Review Program of Moor-FLPI-V2.0 software. The round region of interest (ROI) with the same area in each LSP image was selected for measuring the pancreatic blood perfusion.

Statistics: All findings shown have been reproduced in at least two independent experiments. Data are presented depending on their scale and distribution with arithmetic mean and standard deviation (mean ± s.e.m.). Normality was assessed using a Kolmogorov-Smirnov test. To compare independent measurements we used a t-test and Mann-Whitney U-test as appropriate.

To compare dependent measurements we used a paired t-test. To compare more than two groups we used one-way ANOVA followed by Tukey's post hoc test. Diabetes-free curve is visualized by Kaplan-Meier curves and statistically compared by log-rank test. Statistical analysis was performed using GraphPad Prism v.6. The p values <0.05 were considered statistically significant.


Alexis-Pierre Bemelmans

Sébastien Bonnel

Leila Houhou

Noëlle Dufour

Emeline Nandrot

Dominique Helmlinger

Chamsy Sarkis

Marc Abitbol

Jacques Mallet

Leïla Houhou

Retinal cell type expression specificity of HIV-1-derived gene transfer vectors upon subretinal injection in the adult rat

Keywords: gene transfer, lentiviral vector, pseudotyping, promoter, retinal pigment epithelium, photoreceptors

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

Eye diseases are becoming one of the most promising targets for gene therapy. Various eye diseases, and in particular those involving a neurodegeneration of the retina, fulfill numerous conditions required for "proof of concept" testing for therapeutic gene transfer: (i) animal models of retinal disease allow validation of therapeutic strategies; (ii) several therapeutic genes have been identified and understanding of retinal physiopathology is continuing to improve; (iii) ocular gene transfer needs only local delivery; (iv) existing vitreal-retinal surgical techniques are available to deliver gene transfer vectors to a precise location in the human eye.

For many diseases, particular therapeutic genes will have to be targeted to specific cell types in the retina. In the case of a retinitis pigmentosa caused by a mutation in a gene expressed in photoreceptor cells (PRs), a restorative therapy, in which a fully functional copy of the gene is expressed in PRs, may be possible. Alternatively, protective therapy could be developed based on a factor secreted by the retinal pigment epithelial cells. In both cases a vector that is able to drive transgene expression in specific cell types in the retina is required.

Adenovirus, adeno-associated virus (AAV) and lentivirus-derived vectors have been widely used to transfer genes to retinal cells. Subretinal injections of adenoviral vector lead to efficient transduction of the retinal pigment epithelium (RPE) and only limited transduction of the PRs [START_REF] Bennett | Adenovirus vector-mediated in vivo gene transfer into adult murine retina[END_REF][START_REF] Li | In vivo transfer of a reporter gene to the retina mediated by an adenoviral vector[END_REF][START_REF] Ribeaudeau | In vivo adenovirus-mediated gene transfer to newborn rat retinal pigment epithelial cells[END_REF]. However, adenoviral vectors trigger an immune response which leads to the rejection of the transduced cells [START_REF] Hoffman | Cell-mediated immune response and stability of intraocular transgene expression after adenovirus-mediated delivery[END_REF][START_REF] Reichel | Immune responses limit adenovirally mediated gene expression in the adult mouse eye[END_REF]. Even the last generation of adenoviral vectors, totally devoid of viral coding sequences, do not escape from a transient immune response on administration [START_REF] Thomas | Peripheral infection with adenovirus causes unexpected longterm brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases[END_REF]. This last generation of adenoviral vectors allows transgene expression for longer times than the previous generations, but expression remains transient [START_REF] Kumar-Singh | Encapsidated adenovirus mini-chromosomemediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration[END_REF]. Studies in animals suggest that lentiviral and AAV vectors are more likely to fulfill the criteria for future clinical trials [START_REF] Flannery | Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus[END_REF][START_REF] Miyoshi | Stable and efficient gene transfer into the retina using an HIVbased lentiviral vector[END_REF][START_REF] Bennett | Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina[END_REF]. AAV have been extensively used to transduce retinal cells after subretinal injections. According to the serotype used, they transduce PRs only or PRs and the RPE with a high efficiency [START_REF] Flannery | Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus[END_REF][START_REF] Ali | Gene transfer into the mouse retina mediated by an adeno-associated viral vector[END_REF][START_REF] Bennett | Real-time, noninvasive in vivo assessment of adeno-associated virus-mediated retinal transduction[END_REF]. AAV-mediated gene transfer to the eye appears stable, although one study reported latency in transgene expression that can last between one and several months in primates [START_REF] Bennett | Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina[END_REF]. This delay might be a serious hurdle for clinical applications requiring rapid treatment. Once their effectiveness in transducing CNS cells in vivo was demonstrated, lentiviral vectors were used for the eye [START_REF] Miyoshi | Stable and efficient gene transfer into the retina using an HIVbased lentiviral vector[END_REF], and they transduce the RPE with a high efficiency. Nevertheless, their ability to transfer genes to PRs remains unclear, with some studies demonstrating efficient transduction and others reporting poor or no transduction [START_REF] Auricchio | Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model[END_REF][START_REF] Bainbridge | In vivo gene transfer to the mouse eye using an HIV-based lentiviral vector; efficient long-term transduction of corneal endothelium and retinal pigment epithelium[END_REF][START_REF] Lotery | Gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors[END_REF][START_REF] Duisit | Five recombinant simian immunodeficiency virus pseudotypes lead to exclusive transduction of retinal pigmented epithelium in rat[END_REF].

There are two main strategies currently under investigation for targeting transgene expression to specific cell types. One consists in using an expression cassette containing a cellular specific promoter. This approach has been used with success in various viral vectors, including adenoviruses and lentiviruses [START_REF] Miyoshi | Stable and efficient gene transfer into the retina using an HIVbased lentiviral vector[END_REF][START_REF] Kostic | Activity analysis of housekeeping promoters using self-inactivating lentiviral vector delivery into the mouse retina[END_REF]. The second strategy is to modify the viral vector to transduce cells not naturally infected by the virus. In the case of lentiviruses the possibility of pseudotyping the virus by an envelope other than the widely used vesicular stomatitis virus G protein (VSV-G) protein has recently emerged as a powerful tool to expand the vector tropism to diverse cell types (reviewed in [START_REF] Sanders | No false start for novel pseudotyped vectors[END_REF]).

We report here an investigation of whether HIV-1 derived lentiviral vectors can be engineered to target different cell types of the rat retina, using various combinations of specific promoters and pseudotypes. Recombinant lentiviruses encoding green fluorescent protein (GFP) under the transcriptional control of various ubiquitous and cellular specific promoters were constructed and produced. They were pseudotyped with the classical VSV-G or the lyssavirus Mokola G protein (Mok-G). After subretinal injection into adult rdy rats, GFP expression was analysed to assess the influence of promoters and pseudotyping on lentiviral gene transfer to rat retinal cells. We found that only the combination of the VSV-G pseudotype with the cellular specific rhodopsin promoter allowed efficient transgene expression in few PRs.

Contrarily, RPE cells were very efficiently transduced with both VSV-G and Mok-G.

EXPERIMENTAL METHODS

Lentiviral vectors construction and production.

Plasmids containing the HIV-1 derived vector genome were all constructed from pTrip-CMV-GFP (described in [START_REF] Zennou | HIV-1 genome nuclear import is mediated by a central DNA flap[END_REF]) by replacing the CMV promoter by either the mouse phosphoglycerate kinase 1 promoter (PGK, described in [START_REF] Naffakh | Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts[END_REF]), a 234-bp fragment (ranging from -203 to +31) of the elongation factor 1a promoter (EF1a, described in [START_REF] Sirven | Enhanced transgene expression in cord blood CD34(+)-derived hematopoietic cells, including developing T cells and NOD/SCID mouse repopulating cells, following transduction with modified trip lentiviral vectors[END_REF]) or a 2.2-kb fragment of the human rhodopsin promoter (RHO, amplified from clone pJHN7, kindly provided by Dr J. Bennett, [START_REF] Nathans | Isolation and nucleotide sequence of the gene encoding human rhodopsin[END_REF]). In all cases, the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was added 3' to the GFP gene to increase transgene expression [START_REF] Zufferey | Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors[END_REF]. Vector stocks were produced by transient transfection of 293T cells with the vector genome plasmid, a packaging plasmid and an envelope-coding plasmid as described previously [START_REF] Zennou | HIV-1 genome nuclear import is mediated by a central DNA flap[END_REF]. Briefly, the cell supernatant was harvested 48 hours after transfection, treated with DNase, filtered through 0.45 µm pore-sized filters, and subjected to ultracentrifugation at 64'000 g for 90 minutes. The virus pellet was resuspended in PBS.

Physical titers of the viral stocks were determined by ELISA quantification of the p24 capsid protein concentration using Beckman Coulter kit (Villepinte, France) according to the manufacturer's instructions. All the stocks used in this study had a p24 concentration of the same order of magnitude, ranging from 130 to 250 ng.µl -1 .

Animal care and treatment

Animals were handled in strict accordance with the Helsinsky Declaration and with the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research. The animals were kept at 21°C with a 12-h light:12-h dark cycle and fed ad libitum. All the experiments were performed on young adult (2 to 3 months old) rdy rats (kindly provided by Dr M.M. La Vail, University of California, San Francisco, CA). Subretinal injections were performed under general anaesthesia induced by intra-muscular injection of ketamine/xylazine. Eyes were treated by consecutive local delivery of oxybuprocaine, neosynephrin and tropicamid to inhibit the eyelid reflex and induce pupil dilation. Subretinal injections were given through a trans-scleral approach with a 34G modified Hamilton syringe. The infectious titres of vectors consisting of various promoters and pseudotypes being not comparable, we thus used the amount of p24 protein to define the dose of vector: 100 ng of vector was delivered to each eye in 3 µl of saline containing a small amount of fluorescein. The pressure of injection was set to allow serous detachment without retinal breaks. The injection site was immediately checked by visualisation of the fluorescein position in the fundus under the operating microscope: this allowed differentiating correct subretinal injection from choroidal or intra vitreous injections. Treated and untreated eyes were covered with sterdex paste (Ciba Vision, Basel, Switzerland) containing dexamethasone and oxytetracycline.

In vivo imaging of GFP expression: Fundus Photography and Fluorescein Angiography

In vivo GFP fluorescence was monitored by fundus photography one week and one month after subretinal injection. Rats were anaesthetized by intra-muscular injection of ketamine/xylazine and eyes were treated with neosynephrin and tropicamid to induce pupil dilation. A Canon CF-60ZA human retinal camera modified as previously described was used for fundus photography and fluorescein angiography [START_REF] Diloreto | A new procedure for fundus photography and fluorescein angiography in small laboratory animal eyes[END_REF]. Blue light was used for excitation to detect GFP fluorescence. Then the rats were intraperitoneally injected with 0.15 g/kg of 10% sodium fluorescein (Ciba Vision, Basel, Switzerland) and serial photographs were taken during the 10 min following the injection [START_REF] Shen | Expression of cell adhesion molecules and vascular endothelial growth factor in experimental choroidal neovascularisation in the rat[END_REF] to detect any alteration of retinal tissues induced by the gene transfer and transduction.

Histological procedures

Rats were deeply anesthetized by intra-peritoneal injection of chloralhydrate (500 mg.kg -1 ) and perfused transcardially by ice-cold 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Eyes were then enucleated, and the anterior chamber, cornea, lens and vitreous were discarded. Enucleated eyes were immersed for 2 hours in the same fixative, overnight in PBS containing 30% sucrose, and then in OCT compound immediately before being frozen in dry ice. Serial sections (16 µm) were cut on a cryostat, collected on Superfrost slides, and stored at -80°C until further processing. Alternatively, flat-mounted retinas (after perfusion, dissection and post-fixation) were prepared: neural retina was separated from RPE/choroid/sclera layers and both parts were mounted under coverslips with glycergel.

For immunohistochemical staining of GFP, sections were consecutively incubated in a solution of 5% deionised formamide, 5% 20x saline sodium citrate (20x SSC) and 5% H2O2 in water until depigmentation, in PBS containing 10% normal goat serum (NGS) and 0.2% Triton X-100 for two hours, and overnight in PBS containing 1% NGS, 0.2% Triton X-100 and anti-GFP rabbit polyclonal antibody (ab290, Abcam, Cambridge, UK) diluted 1:5000. Bound primary antibody was then detected according to the manufacturer's instructions with Vectastain ABCkit (Vector laboratories, Burlingame, CA, USA) and using diaminobenzidine (DAB) as the chromogen. Sections were then dehydrated, lightly counterstained with haematoxylin and mounted under coverslips in non-aqueous medium. For immunofluorescence labelling, sections were incubated in PBS containing 10% NGS, 1% gelatine and 0.2% Triton-X100 for two hours, and then overnight in the same solution containing anti-GFP rabbit polyclonal antibody (1:1000 dilution, ab290, Abcam, Cambridge, UK) and anti-GFAP mouse monoclonal antibody (1:300 dilution, GA5, Novocastra Laboratories, Newcastle, UK). Sections were then rinsed three times in PBS, incubated for one hour with secondary antibodies (1:100 FITC-conjugated polyclonal Goat anti-rabbit IgG (H+L), TebuBio, Le Perray en Yvelines, France and 1:300 Cy3-conjugated affinipure goat anti mouse IgG (H+L), Jackson Immunoresearch Laboratories, West Grove, PA, USA), rinsed three times in PBS and mounted under coverslips in Mowiol.

For in situ hybridization, the EGFP coding sequence was inserted into pGEMT-easy (Promega, Charbonnières, France), which was then linearized with NcoI. An antisense riboprobe labelled with digoxygenin (DIG)-UTP was synthesized by the SP6 polymerase and used to probe on the cryostat sections as described in [START_REF] Ravassard | Relax, a novel rat bHLH transcriptional regulator transiently expressed in the ventricular proliferating zone of the developing central nervous system[END_REF]. 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (both from Roche, Meylan, France) were used to obtain a blue staining.

As negative controls, all the histological procedures aiming at detecting the GFP expression have been tested on non-injected eyes and failed to give any signal. Similarly, no direct fluorescence could be detected on flat-mounted retinas of non-injected eyes.

RESULTS

GFP fluorescence was detected in vivo by fundus photography.

We studied GFP expression after subretinal administration of HIV-CMV-GFP pseudotyped with VSV-G or Mok-G. The subretinal location of the injection was assessed by fundus examination. When subretinal injection was successful, subretinal fluids appeared as a precisely delimited bleb with the retinal vessels clearly visible, whereas inadequate intravitreal injection of the vector resulted in a deposit with blurred outlines and masking the retinal vessels (data not shown). For each vector type a total of at least eight correct subretinal injections were performed. One week after injection fundus examination revealed a strong GFP fluorescence in all animals given HIV-CMV-GFP vectors (Figure 1a andb) pseudotyped with either VSV-G or Mok-G. In every case, the area of GFP expression was strictly delimited, and this area was entirely within the subretinal bleb. In some cases, the centre of the transduced area showed no GFP expression, with the most intense fluorescence at the margin of the bleb; this pattern may reflect toxicity associated with the surgery or the vector.

We then tested vectors encoding GFP under the control of house keeping gene promoters (PGK and EF1a) or a cellular specific promoter (RHO). GFP was detected in rats treated with HIV-PGK-GFP and HIV-EF1a-GFP although the signal was lower than with CMV (data not shown). In HIV-RHO-GFP-treated rats, no GFP was detected with either the VSV-G pseudotype or with the Mok-G pseudotype. After testing for GFP, all the animals were injected intra-peritoneally with fluorescein to examine retinal blood vessels and retinal layers (Figure 1c andd). Blood vessels and retinal layers appeared normal in all animals, indicating that subretinal injection of 100 ng of lentiviral vector is not harmful and does not induce a major toxicity event in the retina with reorganization of the various cell layers. Fundus examination one month after vector injection gave the same findings than the examination one week post injection. Thus, irrespective of the pseudotype, vectors with ubiquitous promoters drive strong transgene expression in the retina, whereas GFP was not produced in detectable amounts from the GFP gene under the RHO promoter.

VSV-G and Mok-G pseudotypes transduce the RPE with high efficiency.

We searched for the site of transgene expression. Animals injected with the various vectors were sacrificed one month post injection, after the second fundus examination, and GFP was detected histologically. Firstly, flat-mounted retinas were prepared from two animals of each group to examine direct GFP fluorescence (Figure 2). There was no fluorescence in the neural retina layers with none of the vectors tested as assessed by microscopy. In the RPE/choroid/sclera layers, GFP was detected in eyes that had received HIV-CMV-GFP, HIV-PGK-GFP, or HIV-EF1a-GFP pseudotyped with VSV-G or Mok-G. Higher magnification revealed that the GFP was in cells with typical morphology of the RPE: bi-nucleated and hexagonally shaped (Figure 2c). No fluorescence was detected in flat-mounted retinas of eyes injected with the HIV-RHO-GFP vector stocks. GFP fluorescence was also detected on cryostat tangential sections in an eye injected with a VSV-G pseudotype HIV-CMV-GFP vector (Figure 2d).

VSV-G pseudotype and RHO promoter are necessary to obtain transgene expression in

PRs.

Tangential cryostat sections were prepared from all the remaining eyes and used for immunohistochemistry to determine the site of GFP expression in the retina. In all cases, one month following the transient retinal detachment associated with the subretinal injection, the neural retina had re-attached to the RPE (Figure 3f). We observed a strong staining of the RPE layer in all the eyes injected with ubiquitous promoters containing vectors (HIV-CMV-GFP (n=12), HIV-PGK-GFP (n=11), or HIV-EF1a-GFP (n=8)) (Figure 3). This pattern was independent of the pseudotype, consistent with the findings from flat-mounted retinas (Figure 2). We did not detect any staining in the other retinal layers except in one eye of the Mok-G pseudotyped HIV-PGK-GFP group (n=6, Figure 5c) and one eye of the VSV-G pseudotyped HIV-CMV-GFP group (n=6, Figure 5a). In these two cases, in the middle of the transduced area, GFP immunostaining was evident in the PRs segments, but surprisingly no staining appeared in the corresponding outer nuclear layer (ONL) (Figure 5a andc). In eyes injected with HIV-RHO-GFP vectors, there was a discrete staining of the RPE layer with both VSV-G and Mok-G pseudotypes (Figure 4a andc respectively). In all eyes given VSV-G pseudotyped HIV-RHO-GFP vector (n=8), there were a few PRs that were intensely stained from the nucleus to the segment (Figure 4a). No GFP was found in PRs in eyes given Mok-G pseudotype (n=8). The observations done on retinal sections after immunohistochemical staining of GFP are summarized in table 1.

The RHO promoter displays a weak and ectopic activity in the RPE.

To confirm localization of the transduced cells, we looked for GFP mRNA by in situ hybridization. Consistent with the immunohistochemical staining of GFP, PRs containing GFP mRNA were only found in eyes injected with VSV-G pseudotyped HIV-RHO-GFP (Figure 4b). In these eyes, the colorimetric reaction had to be extended to eight hours to detect GFP mRNA in the RPE layer, confirming the weak activity of the RHO promoter in the RPE (Figure 4b andd). In contrast, an intense staining appeared in the RPE layer after a colorimetric reaction of one hour in eyes injected with HIV-CMV-or HIV-PGK-GFP vectors.

No GFP mRNA was detected in the PRs in these groups even after prolonged reactions (Figure 5b andd).

In the VSV-G pseudotyped HIV-RHO-GFP injected eyes, the cells expressing GFP in the neural retina displayed the characteristic morphology of photoreceptors, with the segments, the nuclei and the synaptic endings clearly visible (Figure 6a andd). No GFP labelling was detected in other retinal layers, except in RPE where some cells were positive, presumably due to an ectopic activity of the RHO promoter (data not shown). To assess for potential inflammation, we performed a labelling of glial fibrillary acidic protein (GFAP), a glial marker up-regulated during inflammation. Close to the injection site, GFAP was up-regulated, as demonstrated by the labelling throughout the cell body of Müller cells, from the ganglion cell layer (GCL) to the ONL (Figure 6b). Further from the injection site, GFAP was detected only close to the GCL (Figure 6e), indicating that GFAP up-regulation was probably due to the surgical act than to transduction of neighbouring cells by the lentiviral vector. No doublelabelled cells were observed, excluding the possibility of Müller cells transduction (Figure 6c and f).

DISCUSSION

We report a study of the tropism of HIV-1 derived lentiviral vectors pseudotyped with the VSV-G envelope or the Mok-G envelope and expressing GFP reporter gene under the transcriptional control of various ubiquitous or cell-specific promoters after injection into the subretinal space of adult rdy rats. Both VSV-G and Mok-G pseudotyped vectors transduced RPE efficiently. In contrast, only VSV-G vectors transduced PRs but the efficiency was poor. Furthermore, PRs were only transduced by constructs carrying the cell-specific rhodopsin promoter, suggesting that the ubiquitous promoters used in this study are not efficient for transgene expression after lentiviral gene transfer in PRs of adult rdy rats.

In our experimental model, the adult rdy rat, VSV-G pseudotyped lentiviral vectors transduced cells of the ONL with a very low efficiency. Few cells were transduced by constructs with the RHO promoter (Figure 4 and6). Substantial GFP immunoreactivity was found in the PR segment layer in two of the thirty-one eyes injected with CMV or PGK vectors but in situ hybridization on adjacent sections indicated that the PRs had not been transduced (Figure 5). The GFP immunostaining observed in the PRs segment layer in these two cases might be the consequence of GFP overproduction in the RPE leading to the presence of GFP in the microvilli of RPE cells or to the release of GFP in the extracellular matrix due to abnormal function of RPE cells.

All studies describing subretinal injections of VSV-G pseudotyped lentiviral vectors encoding reporter genes in rodents or non-human primates report efficient transduction of the RPE, but few demonstrate efficient transduction of the PRs. The discrepancies between studies may be due to the age of the injected animals and the dose of vector used. Miyoshi et al. described GFP expression in RPE and PRs by VSV-G pseudotyped HIV-derived vectors with a CMV and a RHO promoter, and found transduction to be more efficient when rats were injected at 2 to 5 days old than at 4 weeks old, suggesting that maturation of PRs makes them resistant to transduction by VSV-G pseudotyped HIV-derived vectors [START_REF] Miyoshi | Stable and efficient gene transfer into the retina using an HIVbased lentiviral vector[END_REF]. Similarly, Auricchio et al.

described an efficient transduction of the PRs with the same type of vector, but the work was done on 5-7 weeks old mice given large doses of vector [START_REF] Auricchio | Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model[END_REF]. Using a VSV-G pseudotyped FIV vector injected in the subretinal space of cynomolgus monkeys, Lottery et al. showed efficient transduction of the RPE and sporadic transduction of PRs [START_REF] Lotery | Gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors[END_REF], similar to our findings here. Our results are also in accordance with a number of studies showing that in adult rodents, subretinal administration of VSV-G pseudotyped lentiviral vectors preferentially transduce the RPE layer of the retina [START_REF] Bainbridge | In vivo gene transfer to the mouse eye using an HIV-based lentiviral vector; efficient long-term transduction of corneal endothelium and retinal pigment epithelium[END_REF][START_REF] Duisit | Five recombinant simian immunodeficiency virus pseudotypes lead to exclusive transduction of retinal pigmented epithelium in rat[END_REF][START_REF] Takahashi | Rescue from photoreceptor degeneration in the rd mouse by human immunodeficiency virus vector-mediated gene transfer[END_REF][START_REF] Loewen | Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epithelium[END_REF]. Thus, it appears that VSV-G-pseudotyped lentiviral vectors are only suitable for transducing PRs in particular conditions, each of the species, the age, and the dose of vector used might as well be important.

Results obtained by different teams with Mok-G pseudotyped lentiviral vectors are less divergent. Like previous studies [START_REF] Auricchio | Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model[END_REF][START_REF] Duisit | Five recombinant simian immunodeficiency virus pseudotypes lead to exclusive transduction of retinal pigmented epithelium in rat[END_REF], we found that this type of lentiviral vector transduces RPE with an efficiency similar to the one obtained with VSV-G pseudotyped vectors; however these vectors are unable to transduce neuronal cells of the retina. This result is consistent with recent studies showing that after injection into the brain parenchyma, Mok-G pseudotype vectors transduce astroglial cells more efficiently than neuronal cells (Sarkis et al. submitted, [START_REF] Watson | Targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with VSV, Ebola, Mokola, LCMV, or MuLV envelope proteins[END_REF]). Specific promoters could possibly be used instead of pseudotyping to obtain transgene expression restricted to a particular cell type. We compared the activity of ubiquitous promoters with that of the rhodopsin promoter. The rhodopsin promoter drove GFP expression in PRs, as demonstrated by immunostaining and in situ hybridization (Figure 4), but surprisingly, led also to a weak but detectable expression in the RPE. This ectopic expression from the rhodopsin promoter is probably a consequence of a lack of specificity of the fragment used for the genetic construction and of an overload of RPE cells by the vectors, with both the VSV-G and the Mok-G pseudotypes. Kostic et al. described the expression of GFP as being more efficient following transduction of PRs using a VSV-G pseudotyped HIV vector when the subretinal injections were given to newborn mice in comparison to adults [START_REF] Kostic | Activity analysis of housekeeping promoters using self-inactivating lentiviral vector delivery into the mouse retina[END_REF]. Moreover, they report that PRs transduction was favoured by the surgical trauma suggesting that the retinal structure itself may present a physical barrier to transduction of neuronal cells. Thus, it appears that for efficient transduction of rodent PRs with a VSV-G pseudotyped lentivirus, the vector has to be administered very early in life.

The absence of PRs transduction with Mok-G pseudotyped lentiviruses, even, as demonstrated here, with the RHO promoter which is strongly expressed in these cells, demonstrates the value of this pseudotype for prolonged expression of transgenes specifically in RPE cells. An alternate strategy to obtain a transgene expression restricted to RPE cells could be the use of a promoter specific to the RPE, such as the one from the RPE65 gene.

Nevertheless, RPE65 promoter has not yet proven to have an expression strictly restricted in the RPE [START_REF] Znoiko | Identification of the RPE65 protein in mammalian cone photoreceptors[END_REF]. This kind of promoter might be useful for RPE65 gene replacement but Mokola pseudotyping appears currently to be the best strategy for obtaining a transgene expression strictly restricted to RPE cells after lentiviral-mediated gene transfer. This type of vector could be use to correct a genetic defect of the RPE by providing a fully functional gene, or to make these cells function as biological pumps for long-term secretion of a survival factor for the nearby PRs.

LEGENDS

Figure 1 :

 1 Figure 1:

Figure 2 :

 2 Figure 2:

Figure 3 :

 3 Figure 3:

ACKNOWLEDGMENTS

SB is the recipient of a PhD Grant from "Association Française des Amblyopes Unilatéraux". This work was supported by "Association Rétina-France", "Association Française contre les Myopathies", the French "Ministère de la Recherche Programmes Bio-Ingénierie 2000 et 2001", and "Agence Nationale pour la Valorisation de la Recherche" (ANVAR).

Figure 6

vectors. Strong GFP staining was evidenced in every RPE, whereas no GFP was detected in the PRs layer. A: all the neural retina layers are totally devoid of staining. F: photomontage of a retinal section taken at the centre of the eyeball after injection of Mok-G pseudotyped HIV-PGK-GFP. The transduced area, delineated by arrows, covered more than half of the RPE, and, one month after injection, no retinal detachment could be seen. ONL: outer nuclear layer; INL: inner nuclear layer. Scale bar: 20 µm in (A-E), 300 µm in F. Although GFP protein is clearly revealed in the segment layer by immunostaining in both cases (A, C), the in situ hybridization showed that there is no GFP mRNA in the corresponding cells (B, D). Scale bar: 40 µm.

Table 1:

Summary of immunohistochemical results. GFP expression was estimated on both the quantity of expressing cells and the quality of expression in these cells. ++++: strong expression in numerous cells; ++: strong expression in few cells; +/-: weak expression; -: no expression.