Pascale Paul
email: pascale.paul@univ-amu.fr

Romain Boissier

Pauline François

Bastien Gondran Tellier

Maité Meunier

Luc Lyonnet

Stephanie Simoncini

Jeremy Magalon

Tristan Legris

Laurent Arnaud

Laurent Giraudo

Françoise Dignat George

Gilles Karsenty

Stéphane Burtey

Eric Lechevallier

Florence Sabatier

Perirenal Adipose Tissue Displays an Age-Dependent Inflammatory Signature Associated With Early Graft Dysfunction of Marginal Kidney Transplants

Keywords: marginal kidney donors, kidney transplantation, natural killer cells, endothelial inflammation, perirenal adipose tissue, kidney allograft dysfunction

Background: Better understanding of the contribution of donor aging and comorbidity factors of expanded criteria donors (ECD) to the clinical outcome of a transplant is a challenge in kidney transplantation. We investigated whether the features of donor-derived stromal vascular fraction of perirenal adipose tissue (PRAT-SVF) could be indicative of the deleterious impact of the ECD microenvironment on a renal transplant.

Methods: A comparative analysis of cellular components, transcriptomic and vasculogenic profiles was performed in PRAT-SVF obtained from 22 optimal donors and 31 ECD deceased donors. We then investigated whether these parameters could be associated with donor aging and early allograft dysfunction.

Results: When compared with the PRAT-SVF of non-ECD donors, ECD PRAT-SVF displayed a lower proportion of stromal cells, a higher proportion of inflammatory NK cells. The global RNA sequencing approach indicated a differential molecular signature in the PRAT-SVF of ECD donors characterized by the over-expression of CXCL1 and IL1-β inflammatory transcripts. The vasculogenic activity of PRAT-SVF was highly variable but was not significantly affected in marginal donors. Periorgan recruitment of monocytes/macrophages and NK cells in PRAT-SVF was associated with donor aging. The presence of NK cell infiltrates was associated with lower PRAT-SVF angiogenic activity and with early allograft dysfunction evaluated on day 7 and at 1 month post-transplant.

Boissier et al.

Inflammatory Profiles of Marginal Donors

Conclusions: Our results indicate that human NK cell subsets are differentially recruited in the periorgan environment of aging kidney transplants. We provide novel evidence that PRAT-SVF represents a non-invasive and timely source of donor material with potential value to assess inflammatory features that impact organ quality and function.

INTRODUCTION

Increasing recipient demand combined with inadequate organ supply has led to the use of suboptimal marginal kidneys from expanded criteria donors (ECD) with cardiovascular risk factors [START_REF] Ojo | Survival in recipients of marginal cadaveric donor kidneys compared with other recipients and wait-listed transplant candidates[END_REF][START_REF] Rosengard | Report of the Crystal City meeting to maximize the use of organs recovered from the cadaver donor[END_REF][START_REF] Bozkurt | Marginal donors in renal transplantation[END_REF]. While the use of ECD kidney transplants enables more patients to benefit from renal transplantation, various studies have reported that marginal transplants from elderly donors are associated with an increased incidence of delayed graft function (DGF), slow graft function recovery (SGF) [START_REF] Bozkurt | Marginal donors in renal transplantation[END_REF][START_REF] Stevenson | Has the Expansion in extended criteria deceased donors led to a different type of delayed graft function and poorer outcomes?[END_REF][START_REF] Schröppel | Delayed kidney graft function: from mechanism to translation[END_REF](6) and poorer long-term graft outcome [START_REF] Schröppel | Delayed kidney graft function: from mechanism to translation[END_REF][START_REF] Hariharan | Post-transplant renal function in the first year predicts long-term kidney transplant survival[END_REF][START_REF] Audard | Renal transplantation from extended criteria cadaveric donors: problems and perspectives overview[END_REF][START_REF] Siedlecki | Delayed graft function in the kidney transplant[END_REF].

The underlying mechanisms which associate donor age and cardiovascular risk factors with a worsened outcome of these marginal transplants are not completely understood. Since graft endothelial cells constitute the critical interface between the donor and the recipient, pre-existing endothelial dysfunction of the donor could be considered as an initial checkpoint leading to deleterious recipient immune responses and vascular rejection [START_REF] Reinders | Angiogenesis and endothelial cell repair in renal disease and allograft rejection[END_REF][START_REF] Todorova | Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells[END_REF][START_REF] Bussolati | Contribution of stem cells to kidney repair[END_REF][START_REF] Sagban | Vascular challenges in renal transplantation[END_REF]. Several studies have highlighted the "higher immunological risk" of transplants from marginal donors resulting in stress-induced senescence mechanisms [START_REF] Huang | Understanding trends in kidney function 1 year after kidney transplant in the United States[END_REF][START_REF] Schaenman | Differences in gene expression in older compared with younger kidney transplant recipients[END_REF] and induction of endothelial adhesion and inflammatory molecules [START_REF] Danovitch | Current status of kidney and pancreas transplantation in the United States, 1994-2003[END_REF][START_REF] Perico | Long-term clinical and immunological profile of kidney transplant patients given mesenchymal stromal cell immunotherapy[END_REF][START_REF] Xu-Dubois | Markers of endothelial-to-mesenchymal transition: evidence for antibody-endothelium interaction during antibodymediated rejection in kidney recipients[END_REF][START_REF] Dragun | Prolonged cold preservation augments vascular injury independent of renal transplant immunogenicity and function[END_REF].

The current challenge is therefore to delineate the donorrelated features that determine the capacity of transplant endothelium to resist further exposure to ischemia, oxidative, uremic and alloimmune inflammatory stresses associated with the transplant procedure. There is a lack of models that identify donor-related features that reflect the endothelial quality of aging ECD transplants [START_REF] Danovitch | Current status of kidney and pancreas transplantation in the United States, 1994-2003[END_REF].

Our study is based on the hypothesis that perirenal adipose tissue (PRAT), systematically discarded during the surgical preparation of a renal transplant, represents an easily accessible source of donor-derived material allowing assessment of the quantitative and functional features that characterize exposure of donor cells to the ECD microenvironment.

Indeed, adipose tissue (AT) can be enzymatically processed to yield stromal vascular fraction (SVF), a heterogeneous cellular mixture devoid of adipocytes that recapitulates the variety of cells that constitute the vasculature such as mesenchymal stem cells, pericytes, endothelial progenitor cells and leucocytes. Endothelial progenitor cells, also called endothelial colony forming cells Abbreviations: ABM, Agence de la Biomédecine; DGF, delayed graft function; ECD, expanded criteria donors; ECFC, endothelial colony forming cells; GFR, glomerular filtration rate; LD, living donor; NK, Natural Killer cells; Non-ECD, non-expanded criteria donors; PRAT, perirenal adipose tissue; SGF, slow graft function; SVF, stromal vascular fraction.

(ECFC), have been recently identified in the vessel wall and SVF [START_REF] Psaltis | Vascular wall progenitor cells in health and disease[END_REF][START_REF] Lin | Human white adipose tissue vasculature contains endothelial colony-forming cells with robust in vivo vasculogenic potential[END_REF][START_REF] Szöke | Autologous cell sources in therapeutic vasculogenesis: In vitro and in vivo comparison of endothelial colony-forming cells from peripheral blood and endothelial cells isolated from adipose tissue[END_REF]. We and others have reported that the phenotypic and angiogenic activity of ECFC can be altered in deceased contexts associated with cardiovascular risk factors or genetic or epigenetic determinants [START_REF] Aran | Comprehensive analysis of normal adjacent to tumor transcriptomes[END_REF][START_REF] Reutzel-Selke | Donor age intensifies the early immune response after transplantation[END_REF][START_REF] Yazdani | Natural killer cell infiltration is discriminative for antibody-mediated rejection and predicts outcome after kidney transplantation[END_REF]. Furthermore, it was recently demonstrated that donor age and comorbidities can alter the angiogenic and paracrine immunosuppressive properties of human bone marrow-derived stromal cells (BM-SC) obtained after in vitro cell culture expansion [START_REF] Andrzejewska | Multi-parameter analysis of biobanked human bone marrow stromal cells shows little influence for donor age and mild comorbidities on phenotypic and functional properties[END_REF]. It is thus likely that the ECD microenvironment can also alter the vascular potential of the various types of PRAT SVF-resident cells.

Based on this knowledge, we postulated that donor PRAT-SVF could represent a relevant and non-invasive model to evaluate the ECD microenvironment factors that could contribute to the alteration of renal transplant quality. This study aimed to [START_REF] Ojo | Survival in recipients of marginal cadaveric donor kidneys compared with other recipients and wait-listed transplant candidates[END_REF] provide a comprehensive view of cellular, transcriptomic, and angiogenic profiles that could characterize the peri-organ SVF obtained from marginal kidney donors, and (2) analyze whether the features of PRAT-SVF could be indicative of the deleterious impact of donor aging and cardiovascular risk factors on early kidney allograft dysfunction.

MATERIALS AND METHODS

Patients and Sample Collection

We conducted a monocentric prospective study involving 53 renal transplantation procedures performed in the Department of Urology and Renal transplantation, La Conception University Hospital in Marseille, France from 2016 to 2018. For each renal transplant, the stromal vascular fraction (PRAT-SVF) was isolated from the perirenal AT collected during kidney procurement and submitted to analysis of cellular components, transcriptomic profile and vasculogenic activity. The study was approved by the National Ethics Committee of the Agence de la Biomédecine (ABM), the National Ministry of Research and adhered to the Jardé Law on human investigation. All procedures were conducted in compliance with the Declarations of Helsinki and Istanbul. Data were prospectively and anonymously collected in a dedicated database for the exclusive access of the authorized authors.

Clinical Variables

The following demographic data were recorded for donors and recipients: sex, age, body mass index, blood group, serum creatinine, cardiovascular risk factors (history of smoking, hypertension, dyslipidemia, diabetes mellitus, coronary heart disease. Renal function (serum creatinine, glomerular filtration rate) were recorded at D7, M1, and M12 during renal transplantation follow-up. The CKD EPI formula was used to evaluate renal function in adults and the Schwartz formula was used in younger recipients (<18 years) [START_REF] Schwartz | A simple estimate of glomerular filtration rate in children derived from body length and plasma creatinine[END_REF].

Definition of Endpoints

ECD kidney transplants were defined as those from donors aged ≥60 years or 50 to 59 years with 2 of the following comorbidities: hypertension, serum creatinine >1.5 mg/dl, or death following cerebrovascular accident.

Delayed graft function (DGF) was defined as the use of dialysis within 7 days of the transplant [START_REF] Yarlagadda | Marked variation in the definition and diagnosis of delayed graft function: a systematic review[END_REF]. Slow graft function (SGF) was defined by serum creatinine > 250 umol/L (3.0 mg/dL) on postoperative day 7 [START_REF] Dolegowska | Clinical evidence of the association between serum perioperative changes in xanthine metabolizing enzymes activity and early post-transplant kidney allograft function[END_REF].

Identification of Anti-HLA Antibodies

The detection of HLA-specific antibodies was performed using standard techniques. The presence of allograft-specific antibodies was screened through Luminex screening assays (LAScreen R mixed, One Lambda, Canoga Park, CA, USA) using Luminex flow beads (LAScan TM 100, Luminex, Austin, TX, USA). To determine their antibody specificity, all samples with a positive screening result were further evaluated using single-antigen flow bead assays according to the manufacturer's recommended protocol (LAScreen R Single Antigen class I or LAScreen R Single Antigen class II, One Lambda, Canoga Park, CA, USA). The percentage of HLA sensitization for the singleantigen assays were calculated according to the manufacturer's instructions as the percentage of positive bead reactions among the 99 class I beads and 97 class II beads.

Isolation of the Stromal Vascular Fraction From Donor Perirenal AT

Perirenal adipose tissue (at least 30 g) was collected under aseptic conditions during the multi-organ retrieval for cadaveric donor renal transplantation. Excised fat was manually sliced with scissors into units of ∼3 × 3 × 3 mm. Enzymatic digestion was performed using 0.25UI/mL collagenase NB4 (Serva, Heidelberg, Germany) for 1 h at 37 • C under constant agitation. Three cycles of wash/centrifugation were performed to eliminate adipose and red blood cells. Freshly isolated PRAT-SVF was used for flow cytometry analysis and RNA extraction and cryopreserved at -180 • c for delayed angiogenic assays performed on thawed samples.

Phenotypic Characterization of PRAT-SVF Cell Subsets

Multiparameter flow cytometry analysis was performed to compare the quantitative distribution of the CD45-and CD45+ cell subsets in the donor-derived PRAT-SVF in the non ECD and ECD groups.

Half a million cells per tube were suspended in 100 µL of phosphate-buffered saline (PBS, Gibco R , Life Technologies), stained 20 min at room temperature and protected from light with the DRAQ5 nuclear marker, the NucBlue viability marker and two pre-prepared antibody mixes or corresponding isotype controls in matched concentrations. The first monoclonal antibody mix contained the following surface markers: CD146, CD34, and CD45, labeled respectively with the following fluorochromes: PE, ECD, and PC5. The second mix was composed of the following surface markers: CD14, CD34, CD45, CD56, CD3 labeled respectively with FITC, ECD, PC5, PC7 and APC-Alexa Fluor 750 fluorochromes (References in Supplementary Table 1). Flow cytometry was performed with a NAVIOS instrument (Beckman Coulter, Brea, California, USA). Data files were analyzed using Kaluza software (Beckman Coulter, Brea, California, USA). The gating strategy used to identify the various cell subsets is summarized in Supplementary Figure 1.

RNA Purification and RNAseq Gene Expression Analysis

Total RNA was isolated from PRAT-SVF using RNeasy mini kits (QIAGEN Inc., Valencia, CA, USA) including a DNase I digestion step removing genomic DNA. RNAseq analysis of SVF profiles in ECD vs. non-ECD kidney donors was performed by HalioDX. Briefly, the purity and concentration of the samples were estimated by spectrophotometer. The integrity of the RNA (RIN > 8) was evaluated on an RNA 6000 Nano LabChiprun Agilent 2100 Bioanalyzer (Agilent technologies, Germany). Generation of libraries was performed using PerklinElmer technologies with the NEXTflex qRNA-Seq kit v2 after total RNA enrichment by NEXTflex Poly(A) beads following manufacturer's recommendations (PerkinElmer). RNA-seq library were sequenced on Illumina Nextseq sequencer. The generated reads were single-end and of 76-nt length. FastQC (version 0.11.5) was used to examine the read quality. Trimming of reads was performed using Trimmomatic (version 0.33) on the base of an average phred quality of 20. The raw single-ends reads were then mapped against the human genome (GRCh38.90) from the Ensembl database using STAR (version 2.5.3a) sequence mapper. The resulting BAM files were examined by Qualimap (version 2.2). Duplicates reads were removed using the function MarkDuplicates of picard tools (version 2.9.0). Unduplicated reads were used to count reads per gene with FfeatureCounts (version 1.5.2). Raw counts are converted in reads per Million (RPKM) and log transformed (log base 2) in order to help with distributional assumptions, linearity and consistency with PCR based methods for calculating the Fold Change. Genes of interest were filtered using a mean RPKM >25 and a coefficient of variation >50% (1183 genes passed out). R/Bioconductor packages including DESeq2 were used for gene expression analysis. Finally, we selected differentially expressed genes with a P-value <0.05 and a Fold Change (FC) of a least 1.5.

Genes up or downregulated were separately subjected to functional annotation analysis using the Database for Annotation Visualization and Integrated Discovery (DAVID, david.ncifcrf.gov/) online tool to find significantly enriched genes biological functions and associated pathways. Gene Ontology: Biological Process and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was performed with a cut-off criteria for the threshold of EASE score< 0.05 (modified Fisher Exact P-value).

Real Time PCR Analysis of Transcripts

Total RNA (5 µg) was converted to cDNA using 200U of M-MLV reverse transcriptase (Invitrogen). Real-time PCR amplification was performed with the Light Cycler 480 SYBR Green I Master kit (Roche). Cycling conditions were 10 min at 95 • C (hot-start PCR), followed by 40 cycles, 10 s at 95 • C (denaturation), 15 s at 62 • C (annealing) and 20 s at 72 • C (elongation). Melting curve analysis was performed to check the specificity of amplification. Reported values are relative numbers of specific transcripts detected per 10 6 GAPDH transcripts. The primers used for gene-specific amplification are described in Supplementary Table 2.

Tube Formation Assay

Tube formation assay was analyzed in vitro using PRAT-SVF in Matrigel TM (6 mg/ml) (Corning R Matrigel R Basement Membrane Matrix Growth Factor Reduced, Phenol Red Free, 356231) as described by Zakhari et al. [START_REF] Zakhari | Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro[END_REF]. The PRAT-SVF from ECD and non-ECD donors were loaded at a density of 20,000 cells/well in a µ-slide angiogenesis (81506, IBIDI) system coated with 10 µl of growth factor-reduced Matrigel TM (6 mg/ml) (Corning R Matrigel R Basement Membrane Matrix Growth Factor Reduced, Phenol Red Free, 356231), previously polymerized for 30 min, and were maintained in endothelial basal cell culture medium-2 (EBM2) supplemented with MV SingleQuots (EGM2-MV) (Lonza, Clonetics, Walkersville, MD, USA) at 37 • C with 5% CO 2 . Capillary-like structures were recorded after 72 h using a Leica DMI8 video-imaging inverted microscope equipped with an Incubator I8 at 5X magnification and were captured using Leica Application Suite X software (Las X 3.0.2.16120). As previously described [START_REF] Zakhari | Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro[END_REF], various parameters that reflect the relevant steps of SVF vasculogenic/angiogenic capacity in vitro, were quantified: the number of clusters, indicative of the capacity of the plated cells to self-assemble; the number of clusters with tip cells, indicative of the ability of cells to undergo specialization into cells able to migrate away from the cluster and initiate sprouting; the number of clusters with stalk cells that represent the capacity of cells to proliferate and elongate neovessels; and the number of branching points that provide information on the capacity of cells to develop as complex vascular networks. Cell clusters were automatically counted using Fiji software under cellular analysis with minimum object size set at 500 µm and a maximum object size set at 3,000 µm. These counts along with manual counts of tip cells, stalk cells and branch points were taken using still frames of both groups. Each experiment was performed in triplicate.

Spheroid-Based Sprouting Assay

Angiogenic sprouting was analyzed in vitro using PRAT-SVF in a collagen gel matrix as previously described by Korff et al. [START_REF] Korff | Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness[END_REF]. The images were then analyzed using the Sprout Analysis plugin developed by Eglinger et al. [START_REF] Eglinger | Quantitative assessment of angiogenesis and pericyte coverage in human cell-derived vascular sprouts[END_REF] in the Fiji distribution of ImageJ, to evaluate the different vascular parameters, such as sprout length and branch points.

PRAT-SVF from ECD, non-ECD deceased donors and living donors were suspended in culture medium containing 0.2% (wt/vol) carboxymethylcellulose (M0512, Sigma, Munich, Germany), which was then seeded in non-adherent roundbottom 96-well plates (82.1582.001, Sartstedt), leading to the formation of spheroids with a defined cell number. After 72 h, the spheroids were collected and embedded in collagen gels (354236, Corning R Collagen I, Rat Tail). The spheroid containing gel was rapidly transferred into pre-warmed Labtek II slides (NUNC 54534, ThermoFisher) and allowed to polymerize (30 min), then 100 µl of EGM2-MV medium were added on the top of the gel. Following 24 h of culture in EGM2-MV medium, the spheroids were fixed for 30 min in 4% paraformaldehyde at room temperature. After washing and permeabilization 2 h at 4 • C with PBS containing 0.1% Triton X-100 and 1% BSA, the spheroids were immunolabeled overnight at 4 • C with phalloidin coupled with Alexa-647 (A22287, ThermoFisher Scientific) (1/100), and nuclei were stained with 6-diamidino-2-phenylindole (DAPI) (1/5000) diluted in PBS 1% BSA. After washing, we then captured a fluorescent optical image stack along the z-axis at 20X magnification using two lasers in sequential mode under a Leica DMI8 microscope (at least n = 10 spheroids per condition). Las X software was used during all image acquisition procedures. Image processing prior to image measurements was performed with Huygens Essential deconvolution software (Scientific Volume Imaging,) using up to 40 iterations of the classical maximum likelihood estimation algorithm, with a theoretical PSF and automatic background correction. The images were then analyzed using the Sprout Analysis plug-in developed by Eglinger et al. [START_REF] Eglinger | Quantitative assessment of angiogenesis and pericyte coverage in human cell-derived vascular sprouts[END_REF] in the Fiji distribution of ImageJ, to evaluate the different vascular parameters, such as sprout length and branch points.

Statistical Analysis

Statistical analysis comparing continuous variables in 2 groups was performed using non-parametric Wilcoxon-Mann-Whitney test and categorical variables with Chi2 test using Xlstat R version 2018.5 (Addinsoft, Paris, France) and Graphpad Prism R version 7 (GraphPad Software, California, USA). Categorical variables were presented as frequencies and continuous variables as the mean ± standard deviation (SD) or median and 10-90 or 25-75 percentile according to the test of normal distribution using the Kolmogorov-Smirnov test. Spearman rank correlation was used to evaluate the associations between quantitative parameters analyzed in PRAT-SVF and age or creatinine or CKD evaluated in the recipient at D7 and M1 post-transplant. Only variables with P < 0.20 were considered. Significant differences were considered when the P-value was < 0.05. Univariate and multivariate logistic regression analyses were performed to evaluate whether the PRAT-SVF parameters evaluated could discriminate the effect of aging (<59 years) or transplants that had good functional recovery from those with impaired graft function (based on the use of the 60 and 45 ml/ min per 1.73m 2 CKD eGFR cut-off values corresponding to moderate or mild CKD at M1). The AUC of the receiver operating characteristic (ROC) curve was used to define the threshold of quantitative variables that best predicted early dysfunction of the transplant at M1 post-transplant.

RESULTS

Donor and Recipient Characteristics

Fifty-three donors were included: 31 (49%) expanded criteria donors (ECD) and 22 (35%) non-expanded criteria donors (non-ECD). Donor characteristics are summarized in Table 1. Compared with non-ECD, the ECD group presented with higher age (71 vs. 42 years, P < 0.01) and a higher prevalence of cardiovascular risk factors (hypertension, dyslipidemia, vasculopathy, all P < 0.05) except for the prevalence of diabetes that did not reach significance Second transplant concerned 7% of the analyzed cohort. All the patients received the same induction therapy except for one patient in the non ECD donor group that was treated with anti IL2. Rabbit antithymocyte globulin (rATG) were administered on day 0 (1.25 g/kg/day) for 8 days. Prednisolone was administered on day 0 (initially 1 mg/kg/day), with subsequent tapering to achieve a targeted mean maintenance dose of 0.25 mg/kg/day at day 30 after transplant. As previously described [START_REF] Vacher-Coponat | A randomized trial with steroids and antithymocyte globulins comparing cyclosporine/azathioprine versus tacrolimus/mycophenolate mofetil (CATM2) in renal transplantation[END_REF], maintenance immunosuppressive therapy associated Tacrolimus/mycophenolate mofetil (FK/MMF, 64%) or ciclosporine/azathioprine (CSA/Aza, 36%) immunosuppressive combination. All recipients transplanted with an optimal kidney (non ECD) were treated with FK/MMF maintenance therapy, while the CSA/Aza immunosuppressive combination was used in 62 % of the recipients transplanted with an ECD kidney (Table 1).

The recipients of ECD kidney grafts were significantly older than the non-ECD recipients (median 67 vs. 39 years, P < 0.01). However, the prevalence and duration of dialysis before kidney transplantation were comparable in both groups. While most patients were negative for anti HLA panel reactive antibodies (PRA) evaluated before graft (63 %), HLA class I PRA sensitization was observed in 29% of the patients, anti HLA class II sensitization without Class I immunization in 2% of the patients and combined immunization against class I and Class II in 5% of kidney transplant recipients. None of the anti HLA antibodies detected before transplant were Donor specific antibodies (DSA) and transplant recipients analyzed in the study cohort did not develop de novo DSA during the first 3 months following transplant. The incidence of slow/delayed graft function (SDGF) was significantly higher in the recipients of ECD allografts (45% vs. 14%, P = 0.02). The average glomerular filtration rate (eGFR) of ECD donor kidney grafts was significantly lower when compared to non-ECD kidney grafts on day 7 (D7) and at 1 month (M1) post-transplant (with eGFR of 25 vs. 67 ml/min/1.73m 2 on D7 and 41 vs. 82 mL/min/1.73m 2 at M1, all P < 0.05). Mean follow-up post-transplantation period was 12.7 months.

The Cell Subset Distribution of Perirenal SVF Is Altered in ECD Donors

High inter-individual variability in the distribution of SVF cell subsets was observed among the donors (Figure 1). The CD45+ leucocyte population was the most prevalent subset (median 61 %, 25-75 percentile range 47-74) and tended to be higher in the ECD donors (Figure 1A). The median percentage of stromal cells (13%, 25-75 percentile: 7-30%) was significantly lower in the ECD donors (9 %) when compared with the non-ECD donors (18%, p = 0.03) (Figure 1B). The quantitative distribution of endothelial cells (median 8.5 %, 25-75 percentile:

Comparative Analysis of PRAT-SVF the Angiogenic Activity of ECD and Non-ECD Donors

The SVF-dependent formation of capillary-like structures, evaluated in an in vitro Matrigel TM assay (Figure 2A) as the number of clusters, was similar in the ECD and non-ECD donors (Figure 2B). A trend toward a decrease in the tip cell (P = 0.07) as well as stalk cell (P = 0.07) percentages was observed for ECD PRAT-SVF, but did not reach significance (Figure 2B). In addition, the vessel complexity characterized by the Matrigel branching points was also preserved in the ECD donor PRAT-SVF (Figure 2B). In a 3D spheroid assay (Figure 2C), the sprout formation, total network length and the average number of junctions formed by sprouts presented a trend toward a decrease in ECD PRAT-SVF, when analyzed in reference to the non-ECD donor PRAT-SVF (Figure 2D and Supplementary Figure 2). Taken together, these data suggest a high interindividual heterogeneity in the angiogenic potential of donor PRAT-SVF but does not identify a significant impairment of the median angiogenic activity of PRAT-SVF derived from ECD donors, when compared to ECD donors.

Transcriptomic Analysis of PRAT-SVF Identified Inflammatory Profiles Specific to ECD Donors

A comparative RNAseq transcriptomic analysis was performed to compare the PRAT-SVF molecular transcripts in ECD and non-ECD donors (Supplementary Table 3). Volcano plot distinguished a significant differential gene expression profile based on the comparison of ECD and non-ECD patients (Figure 3A). Overall, differential expression analysis revealed 245 genes showing fold change (FC) values ≥ 1.5 (111 genes overexpressed in ECD vs. non-ECD, Supplementary Table 4) and FC ≤ -1.5 (134 genes under-expressed in ECD vs. non-ECD) with P ≤ 0.05 (Supplementary Table 5). To provide a cohesive view of the biological functions associated with the changes in the ECD-SVF gene expression profile, we conducted a gene ontology analysis using the DAVID database. The up-regulated genes showed a strong association with the inflammatory response and cytokine secretion as well as circulatory system development Supplementary Table 6, whereas the categories enriched among the down-regulated genes were associated with the regulation of metabolic processes and the regulation of the circulatory system development (Figure 3B and Supplementary Table 7). Moreover, KEGG pathway analysis revealed differential inflammatory pathways, as "chemokine pathway, " "NF-kappa B pathway" or "TNF signaling pathway, " as well as "Graft-versus-host disease" (Figure 3C and Supplementary Tables 8,9).

Based on their previous involvement in graft rejection or angiogenesis and the FC in their differential expression between ECD and non-ECD patients, five genes were selected (CXCL1, VWA7, CCL4, IL1-β, IFN-γ ) for further quantitative RT-PCR (qPCR) validation in the PRAT-SVF samples used to perform transcriptomic analysis (Supplementary Table 10). Analysis of an extended number of PRAT-SVF samples derived from 12 non-ECD and 13 ECD additional PRAT-SVF samples showed highly variable transcript expression among donors and confirmed the enhanced levels of CXCL1, IL1-β transcripts in ECD donor PRAT-SVF (Figure 3D). Relative transcript levels of CCL4 tended to be higher in ECD PRAT-SVF (p = 0.07, Figure 3D). Thus, these data identified an enrichment of genes involved in the control of inflammatory responses.

Donor Aging Is Associated With Inflammatory Profile in PRAT-SVF

Transcriptomic data prompted further analysis of the distribution of inflammatory cells within CD45+ cell compartment of PRAT-SVF. While the distribution of CD45+CD14+ macrophages/monocytes (Figure 4A), CD45+CD14-neutrophils (Figure 4B), and CD45+CD3+ T lymphocyte subsets (Figure 4C) was comparable among the two groups, the percentage of CD45+CD3-CD56+ NK cells was significantly higher in ECD PRAT-SVF (median value: 2.8%, 25-75 percentile: 1.3-5.1%) compared to non ECD PRAT-SVF (0.97%, 0.4-2.1%, p = 0.01) (Figure 4D). Interestingly, the percentage of NK cells was further associated with the level of transcripts encoding INF-γ inflammatory cytokines and the activating NKG2D receptor (Table 2). Enhanced levels of NK cell infiltrates were also associated with parameters indicative of endothelial dysfunction such as lowered angiogenesis scores and FGFR2 transcript levels (Table 2).

Donor age was also statistically associated with an inflammatory profile characterized by a significantly higher percentage of NK cells in PRAT-SVF. Stratification of donors according to the 59-year median value observed in the cohort confirmed the increased percentage of NK and T cell lymphocytes in the PRAT-SVF of donors ≥59 years (Figure 5). Donor-related factors other than age could not be significantly associated with PRAT-SVF inflammatory profile.

The NK Inflammatory Profile of PRAT SVF Is Associated With Early Allograft Dysfunction

We then investigated whether parameters evaluated in PRAT-SVF could relate to allograft dysfunction during the first month following transplantation. Creatinine levels on D7 and eGFR at M1 post-transplant were significantly correlated with donor age, but did not correlate with cold ischemia time in the studied cohort (Table 3). Early graft dysfunction, as defined by creatinine levels at day 7 and values of eGFR<45 mL/min at one-month (M1) after transplantation, were also correlated with the proportion of PRAT-SVF NK inflammatory cells and monocytes/macrophages (Table 3 and Figure 6) in univariate analysis. We used ROC curve analysis to set a 1.5% NK cell threshold in PRAT-SVF associated with lower CKD at M1 (area under ROC curve = 0.82, sensitivity 100%, specificity 75%). Interestingly, logistic regression models further showed that a percentage of NK cells > to this 1.5 threshold value of NK cells observed in pre-transplant donor PRAT-SVF, was an independent factor associated with lowered graft function recovery (eGFR< 45 or 60 at 1-month post-transplant), regardless of the HTA status of the donor (Table 4).

DISCUSSION

Taking advantage of the accessibility of donor perirenal adipose tissue, our study is the first to evidence significant changes in the cellular and molecular features that characterize the PRAT-SVF of marginal donors. Compared with optimal donors, marginal donors exhibited significant alterations in the cellular composition of PRAT-SVF that notably comprised an increase in immune-cell infiltrates and levels of transcripts encoding inflammatory chemokines/cytokines. This molecular inflammatory signature was impacted by donor age and could be further associated with early graft dysfunction.

The cell subset distribution in PRAT-SVF of ECD transplants indicated an imbalance between pro and anti-inflammatory cells. ECD donor PRAT-SVF showed a lowered proportion of stromal/mesenchymal cells, recently identified as an immunomodulatory cell compartment of the perirenal adipose tissue [START_REF] Baer | Isolation, characterization, differentiation and immunomodulatory capacity of mesenchymal stromal/stem cells from human perirenal adipose tissue[END_REF], and a higher proportion of immune NK lymphocyte infiltrates. Interestingly, enhanced representation of NK and T cells in PRAT-SVF was identified as an age-related specific feature. These results are in line with experimental studies suggesting that kidneys from older donors are more immunogenic and induce increased T-cell responses than kidneys from young donors [START_REF] Reutzel-Selke | Donor age intensifies the early immune response after transplantation[END_REF]. Consistently, major changes in the transcriptomic signature of ECD donors were found to be related to upregulation of inflammatory pathways. Among the most up-regulated genes was CXCL1, which is also known as GROα. CXCL1 is a pro-inflammatory chemokine that binds to CXCR2 to promote neutrophil chemotaxis. CXCL1-dependent neutrophil accumulation in a kidney transplant after reperfusion is an important predictor of delayed graft function [START_REF] Turunen | Association of graft neutrophil sequestration with delayed graft function in clinical renal transplantation[END_REF][START_REF] Stadtmann | CXCR2: From Bench to Bedside[END_REF] . CXCL1 has also been associated with various inflammatory kidney diseases such acute kidney ischemia and glomerulonephritis [START_REF] Van Gemst | Differential binding of chemokines CXCL1, CXCL2 and CCL2 to mouse glomerular endothelial cells reveals specificity for distinct heparan sulfate domains[END_REF] and progression of chronic kidney disease [START_REF] Rudnicki | Renal microRNA-and RNA-profiles in progressive chronic kidney disease[END_REF]. Inhibition of CXCR2 prevents kidney graft function deterioration owing to ischemia/reperfusion [START_REF] Cugini | Inhibition of the chemokine receptor CXCR2 prevents kidney graft function deterioration due to ischemia/reperfusion[END_REF]. Our transcriptional analysis also demonstrated that upregulation of the CCL4 chemokine and IL1beta were associated with the ECD profile. This upregulation was consistent with an enhanced proportion of NK cells infiltrating ECD donor PRAT-SVF and previous data reporting the activation of a CCL4 and IFN-γ dependent pathway in patients with kidney graft rejection [START_REF] Halloran | Review: The transcripts associated with organ allograft rejection[END_REF]. Although extrapolation of these observations in donor PRAT-SVF could not be matched with those occurring in the pre-transplant biopsy, these results corroborate previous findings that identify the NKG2D activating receptor as a candidate marker of kidney graft quality in pretransplant biopsy specimens from donors over 55 years [START_REF] Günther | Identification of the activating cytotoxicity receptor NKG2D as a senescence marker in zerohour kidney biopsies is indicative for clinical outcome[END_REF].

In kidney transplant recipients, innate NK cells have recently been identified as a key effector mechanism regulating the level of endothelial lesion and repair as well as vascular rejection and allograft vasculopathy [START_REF] Todorova | Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells[END_REF][START_REF] Yazdani | Natural killer cell infiltration is discriminative for antibody-mediated rejection and predicts outcome after kidney transplantation[END_REF][START_REF] Parkes | Evidence for CD16a-Mediated NK Cell Stimulation in Antibody-Mediated Kidney Transplant Rejection[END_REF][START_REF] Legris | Antibody-dependent NK cell activation is associated with late kidney allograft dysfunction and the complement-independent alloreactive potential of donor-specific antibodies[END_REF][START_REF] Kildey | Specialized roles of human natural killer cell subsets in kidney transplant rejection[END_REF]. NK cells have also been reported to contribute to immune senescence in kidney transplant candidates [START_REF] Dewolfe | NK cells contribute to the immune risk profile in kidney transplant candidates[END_REF]. Our observations suggest that PRAT-SVF recruitment of donor NK cells could also promote proinflammatory signals that affect the vascular homeostasis of a marginal transplant prior transplantation. While the quantitative distribution of PRAT-SVF endothelial cells was comparable in ECD and non-ECD donors, we observed a high inter-individual variability in PRAT-SVF angiogenic activity, that did not reach significance between the ECD and non-ECD groups analyzed in the present study. Such heterogeneity among donors has already been reported for mesenchymal stem cells [START_REF] Andrzejewska | Multi-parameter analysis of biobanked human bone marrow stromal cells shows little influence for donor age and mild comorbidities on phenotypic and functional properties[END_REF]. However, in line with this result, the study by Aird et al. did not evidence major changes in the angiogenic potential of AT-SVF with aging except a delayed phase of neovessels maturation in vivo [START_REF] Aird | Adipose-derived stromal vascular fraction cells isolated from old animals exhibit reduced capacity to support the formation of microvascular networks[END_REF]. However, we observed that the percentage of NK cells in PRAT-SVF was inversely correlated with the angiogenic potential, suggesting that, at an individual level, donor-dependent NK cell activation could also provide an inflammatory environment that favors endothelial vulnerability prior to transplantation. Importantly, among the parameters analyzed with PRAT-SVF, the proportion of NK cells was identified as associated to graft dysfunction evaluated at 7 days and 1-month post transplantation, indicating a potential impact on the clinical [START_REF] Dewolfe | NK cells contribute to the immune risk profile in kidney transplant candidates[END_REF]. The proportion of cell subsets was analyzed according to this splitting. The eGFR M1 < 45 presented a significantly higher percentage of monocyte/macrophage subset and NK cells. Other cells were similarly distributed between the two groups.

outcome of marginal transplants from aging donors. These findings make it possible to speculate that the heterogeneity of inflammatory cytokine overexpression and age-dependent NK cell activation in ECD transplants could contribute to shaping allograft immunogenicity by perpetuating immune cell recruitment and activation, thereby rendering the endothelial cells of the graft more vulnerable to further exposure to ischemia/reperfusion, uremic, and alloimmune inflammatory stresses. These markers could thus be regarded as potential molecular targets for strategies enabling to reduce inflammation in ECD transplants.

Our study presents limitations since the unavailability of pretransplant renal biopsies did not enable evaluation of the specific features that characterized PRAT-SVF in marginal donors which could be extended to the renal parenchyma [START_REF] Wang | The donor kidney biopsy and its implications in predicting graft outcomes: a systematic review[END_REF]. However, these data provided evidence that tissue recruitment of donor NK cells may per se participate in the pre-conditioning of transplant vulnerability and quality and prompt further investigation of the clinical relevance of such biomarkers in larger cohorts. This work could introduce PRAT-SVF as an innovative and less invasive approach with added value in terms of feasibility compared with pre-implantation biopsies and also document the specific features that characterize perirenal fat [START_REF] Wang | The donor kidney biopsy and its implications in predicting graft outcomes: a systematic review[END_REF][START_REF] Naesens | Zero-time renal transplant biopsies: a comprehensive review[END_REF]. Another limitation is that the RNAseq and the functional analysis of the angiogenic capacity of donor-derived cells were performed on the whole PRAT-SVF and not on individual SVF sorted cell types. This experimental design allowed to integrate the cellular crosstalk between SVF cell subsets and prevented a potential bias associated with cell subset isolation and expansion in vitro. However, it did not allow to define if the observed changes resulted from alteration of PRAT-SVF composition or from a specific imprint of the ECD microenvironment on a given cell type. These data call for a more in-depth analysis using a single cell approach characterizing the transcriptomic profile of PRAT-SVF specific to the microenvironment of the ECD donor and the specific study of mesenchymal and endothelial cells purified from perirenal SVF. Future single cell analysis approaches and comparative analyses of purified endothelial and mesenchymal cells isolated from PRAT-SVF ECD and optimal donors would be of value to provide additional mechanistic clues.

Although the immediate implications of PRAT-SVF are not compatible with the current clinical setting of transplantation, our work may open perspectives to target inflammatory pathways in order to reduce donor-related inflammation before transplantation during the dynamic hypothermic machine perfusion process with the aim to optimize transplant quality.

CONCLUSION

Our results argue in favor of a donor-dependent inflammationdriven alteration of pre-transplant allograft quality and identify NK cells as potential effectors of pro-inflammatory remodeling mechanisms that can affect the function of marginal elderly transplants.

FIGURE 1 |

 1 FIGURE 1 | The distribution of cell subsets composing the PRAT-SVF was determined using flow cytometry and compared between the non-ECD and ECD donors: (A) CD45 + leukocytes were comparable in the two groups (B) mesenchymal stem/stromal cells were significantly lowered in the ECD vs. non-ECD group (C) pericytes and transitional cells (D) and endothelial cells were not statistically different between the two groups. ECD, extended criteria donors. Non ECD, non-extended criteria donors. Results on the graphs are reported as box and whiskers plots representative of median values, and 25-75 interquartile ranges (Boxes) and error bars indicative of 10-90 percentile ranges. Dots indicates values out of the 10-90% quartile range.

FIGURE 2 |

 2 FIGURE 2 | (A) Representative experiment of capillary tube formation by SVF from ECD or non-ECD. A total of 20,000 cells/well were seeded on growth factor reduced Matrigel. Images were recorded at 72 h with a phase-contrast microscope. Original magnification x5; upper panel (scale bars, 300 µm) correspond to the total image of the well while the lower panels were zooms of previous images (scale bars, 200 µm). White arrows identify initial cell clustering; red arrows marked the tip cells while yellow arrows identified branching. (B) Quantitative analysis of number of clusters, percentage of clusters with tip and stalk cells, number of branching points. Data are expressed as means ± SEM of independent experiments performed in triplicate using PRAT-SVF obtained in 14 ECD and 10 non-ECD donors (C) Representative experiment of 3D in vitro angiogenic assay with collagen gel-embedded spheroids of SVF from ECD or non-ECD (original magnification x20; scale bars, 100 µm). Imaging of Vascular sprouts was obtained after merging of actin staining (phalloidin in green) and nuclei staining (DAPI, blue) as detailed in Supplementary Figure 2 (D) Quantitative analysis of number of sprouts, branch points, and total network length per spheroid as well as average sprout length was compared in PRAT-SVF from the ECD and non ECD donors. For each experiment, at least 10 spheroids were analyzed.

FIGURE 3 |

 3 FIGURE 3 | Comparative Transcriptomic analysis of SVF from ECD (n = 5) and non-ECD (n = 5) patients. (A) Volcano plot of differentially expressed genes from SVF from ECD and non-ECD. Log2 Fold Change value obtained by RNAseq plotted against the -log10 of P-value. Genes with a fold change > |1.5| and P < 0.05 were deemed to be differentially expressed. P = 0.05 is indicated by horizontal lines. Positive and negative fold change values are reflective of down-regulated (green) or up-regulated (red) genes compared with non-ECD condition, respectively. (B) Fold enrichment over chance for the Gene Ontology Biological process of the Down (gray) and Up (black) gene lists using DAVID (fold change > |1.5| and P < 0.01). (C) Fold enrichment over chance for the KEGG Pathway of the Down (gray) and Up (black) gene lists using DAVID (fold change > |1.5| and P < 0.01). (D) qRT-PCR validation of selected genes expressed a relative levels of specific transcripts detected per 10 6 GAPDH transcripts [median (25-75% quartile)].

FIGURE 4 |

 4 FIGURE 4 | The distribution of CD45 + hematopoietic cells composing the PRAT-SVF of ECD and non-ECD donors was analyzed using flow cytometry according to the gating strategy illustrated in Supplementary Figure 1. Results on the graphs are reported as box and whiskers plots representative of median values, and 25-75 interquartile ranges (Boxes) and error bars indicative of 10-90 percentile ranges. (A) CD14+ Monocyte/Macrophage subset. (B) CD14-neutrophil subset. (C) CD3+ T lymphocytes. (D) CD3-CD56+ NK cells. (E) Histograms illustrate the gating and % of CD3+ T cells and CD3-CD56+ NK Cells observed when analyzing PRAT-SVF of a non-ECD donor (0.43% of NK Cells) or (F) PRAT-SVF from an ECD donor (9.47% of NK cells).

FIGURE 5 |

 5 FIGURE 5 | Analysis of perirenal adipose tissue stromal vascular fraction (PRAT-SVF) in young vs. aged donors. PRAT-SVF was analyzed according to donor age when stratified in aging donors (≥59 years, n = 29) and younger donors (<59 years, n = 24). Percentages in stromal and endothelial cells were not different between the aging and younger donors. However, the aging donors presented a trend for increased representation of the CD45+ CD14+ monocyte macrophage cell subset and a significantly higher percentage of T and NK cells.

FIGURE 6 |

 6 FIGURE 6 | Analysis of perirenal adipose tissue stromal vascular fraction (PRAT-SVF) with lower recovery of graft persisting at M1 post-transplant. Donors were split into two groups according to graft recovery persisting at M1 post-transplant: lower recovery (eGFR M1 < 45) and normal recovery (eGFR >[START_REF] Dewolfe | NK cells contribute to the immune risk profile in kidney transplant candidates[END_REF]. The proportion of cell subsets was analyzed according to this splitting. The eGFR M1 < 45 presented a significantly higher percentage of monocyte/macrophage subset and NK cells. Other cells were similarly distributed between the two groups.

TABLE 1 |

 1 Donors and recipients characteristics.

	ECD	Non-ECD P-value
	n = 31	n = 22

TABLE 2 |

 2 Analysis of the link between quantitative parameters evaluated in PRAT-SVF and % of PRAT-SVF NK cells.

	% CD3-CD56+ NK cell PRAT-SVF	Spearman r	P-value
	DONOR CHARACTERISTICS
	Donor age	0.6228	0.0007***
	PRAT SVF IMMUNE CELLS
	% CD3+ T cell PRAT-SVF	0.4845	0.0121*
	% CD14+ Mono/macro PRAT-SVF	0.4216	0.0319*
	PRAT-SVF TRANSCRIPTS
	PRAT-SVF NKG2D transcript Levels	0.676	0.0021**
	PRAT-SVF FGFR2 transcript levels	-0.624	0.0098**
	PRAT-SVF IFN-γ transcript levels	0.5611	0.0101*
	ANGIOGENESIS
	Spheroid total network length	-0.5368	0.0178*
	Spheroid average junction per sprout	-0.5035	0.028*
	Spheroid number of sprouts	-0.4906	0.033*
	Matrigel number of clusters	-0.4719	0.0413*

BMI, Body Mass Index; PRAT, PeriRenal Adipose Tissue; SVF, Stromal Vascular Fraction. *P < 0.05, **P < 0.01, ***P < 0.001.

TABLE 3 |

 3 Analysis of parameters correlating with early graft function at Day 7 (D7) and 1 month (M1) post-transplant.

		Serum creatinine Day 7	eGFR M1
	PRAT-SVF donor	Spearman r P-value	Spearman r P-value
	DONOR CHARACTERISTICS
	Donor Age	0.3725	0.006	-0.5371	0.0001
	Cold Ischemia time (hours)	0.2666	0.0802	ns	ns
	PRAT-SVF IMMUNE CELLS INFILTRATION
	% CD45+CD14+ Monocytes/	0.4461	0.0289	-0.4874	0.0214
	macrophages
	% CD3-CD56+ NK cells	0.3991	0.0533	-0.4485	0.0363
	% CD3+ T cells	0.34	0.104	ns	ns
	PRAT, PeriRenal Adipose Tissue; SVF, Stromal Vascular Fraction; GFR, Glomerular
	Filtration Rate; Ns, Non-significant.

TABLE 4 |

 4 Logistic regression models linking the presence of PRAT-SVF NK cell infiltrates to lowered allograft function evaluated 1-month (M1) post-transplantation.

		Odds Ratio Std. Err.	z	p	[95% Conf. Interval]
	Predictive factors of CKD<45 mL/min M1
	NK SVF ≥1.5%	25.6	33.5	2.48 0.01	[1.9-332.4]
	Donor HTA	6.7	10.1	1.26 0.21	[0.3-130.2]
	Predictive factors of CKDM1<60 mL/min M1
	NK SVF ≥ 1.5%	10.6	11.2	2.2 0.03	[1.3-84.8]
	Donor HTA	4.4	5.9	1.1 0.27	[0.3-61.7]

Frontiers in Immunology | www.frontiersin.org

March 2020 | Volume 11 | Article 445

ACKNOWLEDGMENTS

The authors would like to thank the tissue donors and clinicians, particularly urologists, and nephrologists that contributed to collection of peri-renal adipose tissue.

FUNDING

This project was funded in part by the Fondation de l'Avenir, APM 2015 Paris, France and the Association Française d'Urologie, Paris, France and INSERM.

DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. The RNAseq data has been submitted to the GEO repository under accession numbers GSE140122.

ETHICS STATEMENT

The study was approved by the National Ethics Committee of the Agence de Biomédecine (ABM), the National Ministry of Research and adhered to the Jardé Law on human investigation. All procedures were conducted in compliance with the Declarations of Helsinki and Istanbul. Data were prospectively and anonymously collected in a dedicated database for the exclusive access of the authorized authors.

AUTHOR CONTRIBUTIONS

RB, PP, GK, EL, and FS contributed to the conception and design of the study. RB, GK, MM, EL, BG, TL, and SB enrolled subjects into the study, collected primary data. LL, PF, BG, SS, JM, LG, and LA performed the experiments. PP reviewed the statistical analysis and wrote the manuscript. FD and FS wrote sections of the manuscript. All authors contributed to manuscript revision and read and approved the submitted version.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.00445/full#supplementary-material Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vasilis Petropoulos

Lorenzo Uboldi

Margherita Maiuri

Giulio Cerullo
email: giulio.cerullo@polimi.it

Lara Martinez-Fernandez

Evangelos Balanikas

Dimitra Markovitsi
email: dimitra.markovitsi@universite-paris-saclay.fr

Lara Martinez- Fernandez

Effect of the DNA Polarity on the Relaxation of Its Electronic Excited States

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

The polarity of nucleic acids, that is the order according to which two adjacent nucleobases are linked together within a strand via the phosphodiester backbone, from the 5' end to the 3'end, [START_REF] Alberts | The Structure and Function of DNA[END_REF] determines the adopted conformations, [START_REF] Hazel | Loop-length-dependent folding of G-quadruplexes[END_REF][START_REF] Bansal | A short GC-rich palindrome of human mannose receptor gene coding region displays a conformational switch[END_REF][START_REF] Sket | Influence of 3 '-3 ' inversion of polarity site within d(TGGGGT) on inter quartet cation binding[END_REF] and play a key role in biological functions depending on structural features. [START_REF] Gupta | Polarity of DNA strand exchange promoted by recombination proteins of the RecA family[END_REF][START_REF] De Laat | DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair[END_REF][START_REF] Balasingham | Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB[END_REF][START_REF] Lin | A 5-to-3 strand exchange polarity is intrinsic to RecA nucleoprotein filaments in the absence of ATP hydrolysis[END_REF] Among others, it has been reported that the DNA/RNA strand polarity has an influence on processes involving various redox reactions, [START_REF] Saito | Photoinduced DNA Cleavage via Electron Transfer -Demonstration that Guanine Residues located 5' to Guanine are the Most Electron-donating sites[END_REF][START_REF] Virgilio | Improved performances of catalytic G-quadruplexes (G4-DNAzymes) via the chemical modifications of the DNA backbone to provide G-quadruplexes with double 3 '-external G-quartets[END_REF][START_REF] Cao | Manipulating the Assembly of DNA Nanostructures and Their Enzymatic Properties by Incorporating a 5 '-5 ' Polarity of Inversion Site in the G-Tract[END_REF][START_REF] O'neill | Effects of strand and directional asymmetry on base-base coupling and charge transfer in double-helical DNA[END_REF] as well as hole transport in DNA. [START_REF] Voityuk | Electronic coupling between Watson-Crick pairs for hole transfer and transport in desoxyribonucleic acid[END_REF] A transfer of an electrical charge takes also place between stacked nucleobases following absorption of UV radiation by DNA, leading to population of excited charge transfer (CT) states. Femtosecond transient absorption studies showed that, in DNA multimers, an important part of the initially excited state population evolves toward CT states. [START_REF] Chen | Excited states in DNA strands investigated by ultrafast laser spectroscopy[END_REF][START_REF] Schreier | Early Events of DNA Photodamage[END_REF][START_REF] Kwok | Bright" and "Dark" excited states of an alternating AT oligomer characterized by femtosecond broadband spectroscopy[END_REF][START_REF] Keane | Long-lived excited states in i-motif DNA studied by picosecond time-resolved IR spectroscopy[END_REF][START_REF] Doorley | Tracking DNA Excited States by Picosecond-Time-Resolved Infrared Spectroscopy: Signature Band for a Charge-Transfer Excited State in Stacked Adenine-Thymine Systems[END_REF][START_REF] Doorley | Picosecond transient infrared study of the ultrafast deactivation processes of electronically excited B-DNA and Z-DNA forms of [poly(dG-dC)] 2[END_REF][START_REF] Ma | Real-time Monitoring Excitation Dynamics of Human Telomeric Guanine Quadruplexes: Effect of Folding Topology, Metal Cation, and Confinement by Nanocavity Water Pool[END_REF][START_REF] Kwok | Femtosecond time-and wavelength-resolved fluorescence and absorption study of the excited states of adenosine and an adenine oligomer[END_REF][START_REF] Kufner | UV-Induced Charge-Transfer States in Short Guanosine-Containing DNA Oligonucleotides[END_REF] The latter survive much longer, up to several hundreds of ps, while the monomer excited states decay within, at most, 10 ps.

Theoretical studies predicted that CT states should be affected by the strand polarity, [START_REF] Colon | Role of sequence and conformation on the photochemistry and the photophysics of A-T DNA dimers: An experimental and computational approach[END_REF][START_REF] Santoro | The excited electronic states of adenineguanine stacked dimers in aqueous solution: a PCM/TD-DFT study[END_REF][START_REF] Aquino | The Charge-Transfer States in a Stacked Nucleobase Dimer Complex: A Benchmark Study[END_REF][START_REF] Martinez-Fernandez | Novel adenine/thymine photodimerization channels mapped by PCM/TD-DFT calculations on dApT and TpdA dinucleotides[END_REF][START_REF] Martinez-Fernandez | Sequence dependence on DNA photochemistry: a computational study of photodimerization pathways in TpdC and dCpT dinucleotides[END_REF][START_REF] Lee | Role of charge transfer states into the formation of cyclobutane pyrimidine dimers in DNA[END_REF][START_REF] Balanikas | The Structural Duality of Nucleobases in Guanine Quadruplexes Controls Their Low-Energy Photoionization[END_REF] which determines the relative orientation of the stacked nucleobases and, hence, the associated orbital overlap (Figure 1a). Here, focusing on dinucleotides composed of adenine and guanine, we provide the first experimental evidence for such an asymmetrical behavior. We show that the spectral and dynamical features determined for 5'-dApdG-3' (abbreviated as AG) differ from those of 5'-dGpdA-3' (abbreviated as GA), the latter configuration favoring the formation of CT states and slowing down their decay rate. These observations are supported by quantum chemistry computations performed for the same systems.

AG and GA, purified by reversed phase HPLC and tested by MALDI-TOF (Figure S1 in the Supporting Information, SI), were purchased from Eurogentec. They were dissolved in high concentration phosphate buffer (0.12 molL -1 , pH 7.0), so as to promote stacking, and subsequently excited at 266 nm (Figure 1b). Their transient absorption spectra were recorded from 330 to 650 nm for delays reaching 50 ps by means of a homemade pump-probe setup (I) with a time resolution of 30 fs, already used for the study of DNA. [START_REF] Borrego-Varillas | Exciton Trapping Dynamics in DNA Multimers[END_REF] The long-time decays were determined in the visible by means of a second setup (II) allowing measurements till 1.2 ns with a time resolution of 100 fs, after checking that the signals obtained with both setups overlap in the 0.1 -50 ps range (Figure S2). To avoid photodegradation, 6 mL of the solution were kept flowing through a quartz cell whose optical path-length was 1 mm. The concentration of absorbed photons (8×10 -6 molL -1 ) was much lower than the dinucleotide concentration (2-3×10 -4 molL -1 ), making two-photon excitation highly improbable. More experimental details are given in the SI. The dinucleotide steady-state absorption spectra are slightly blue-shifted with respect to that of an equimolar mixture of the mononucleotides dAMP and dGMP [START_REF] Onidas | Fluorescence properties of DNA nucleosides and nucleotides: a refined steady-state and femtosecond investigation[END_REF] (Figure 1b), suggesting some chromophore interaction in the ground state. Thus, their time-resolved behavior should result from an ensemble of configurations, ranging from unstacked chromophores to fully stacked ones.

In the limiting case of non-interacting nucleobases, monomer-like decays are expected. The transient absorption spectra of the adenosine and guanosine chromophores are shown in Figure 1c. The former, [START_REF] Kwok | Femtosecond time-and wavelength-resolved fluorescence and absorption study of the excited states of adenosine and an adenine oligomer[END_REF][START_REF] Borrego-Varillas | Exciton Trapping Dynamics in DNA Multimers[END_REF] exhibits two well defined peaks, at 375/380 and 640/685 nm. [START_REF] Doorley | Picosecond transient infrared study of the ultrafast deactivation processes of electronically excited B-DNA and Z-DNA forms of [poly(dG-dC)] 2[END_REF][START_REF] Lee | Role of charge transfer states into the formation of cyclobutane pyrimidine dimers in DNA[END_REF] In contrast, only one intense band, peaking around 300 nm, is present in the latter. [START_REF] Karunakaran | Photoinduced dynamics of guanosine monophosphate in water from broad-band transient absorption spectroscopy and quantum-chemical calculations[END_REF][START_REF] Cheng | The solvent effect and identification of a weakly emissive state in nonradiative dynamics of guanine nucleosides and nucleotides -a combined femtosecond broadband time-resolved fluorescence and transient absorption study[END_REF] Adenosine decays much faster than guanosine, with the time needed for the intensity of their UV transient absorption band to decrease by a factor of 10 being, respectively, 1 and 8 ps. [START_REF] Borrego-Varillas | Exciton Trapping Dynamics in DNA Multimers[END_REF][START_REF] Karunakaran | Photoinduced dynamics of guanosine monophosphate in water from broad-band transient absorption spectroscopy and quantum-chemical calculations[END_REF] The behavior of the AG and GA dinucleotides considered here, in which the nucleobases are stacked in the ground state was rationalized with the help of quantum chemistry calculations in a previous publication. [START_REF] Balanikas | The Structural Duality of Nucleobases in Guanine Quadruplexes Controls Their Low-Energy Photoionization[END_REF] In that theoretical study the entire system (two nucleobases, the connecting backbone and the counter ion), embedded in water through a polarizable continuum model, was taken into account. The relative position of the nucleobases in the ground state of each system, derived from geometry optimization, is depicted schematically in Figure 1a. The stacking distance is comparable in both systems, around 3.2 Å. Computations provided various minima in the potential energy surface of the first excited state. [START_REF] Balanikas | The Structural Duality of Nucleobases in Guanine Quadruplexes Controls Their Low-Energy Photoionization[END_REF] For both systems, a * state (La) localized on the guanine as well as a G   A -CT state were identified, with  being equal to 0.5 a.u. for AG and 0.7 a.u. for GA. [START_REF] Balanikas | The Structural Duality of Nucleobases in Guanine Quadruplexes Controls Their Low-Energy Photoionization[END_REF] Furthermore, a * exciton state, delocalized over the adenine and the guanine, was found only for AG. In the present study, we computed the transient absorption spectra corresponding to the above-mentioned minima (Figures 2a and2b). To this end, we used a multifunctional analyzer (multiwfn program) [START_REF] Lu | A multifunctional wavefunction analyzer[END_REF] to obtain the excited-excited state transition dipole moments. Additional computational details are provided in the SI. The spectra computed for the * localized excited states are characterized by an intense peak in the UV, a shoulder around 400 nm and a weak intensity band peak above 600 nm. Despite these similarities, subtle differences among the spectra of the two systems can be observed: the UV band of AG is slightly red-shifted ( max = 338 nm) compared with that of GA ( max = 312 nm), while the red band is more intense for the former system. The spectrum of the AG exciton is very broad, extending all over the examined spectral domain, its intensity peaking in the 400 -450 nm region. Regarding the spectra of the CT states, the UV band is more intense for AG and is located at shorter wavelengths (337 nm vs 367 nm) compared to the one of GA; in addition, the AG band in the visible is less intense and blue shifted with respect to that of AG. The spectra of the CT states, normalized at their maximum, are compared in Figure 2c.

Based on the observations on the excited state relaxation of the non-interacting adenosine and guanosine chromophores in neutral aqueous solution, [START_REF] Kwok | Femtosecond time-and wavelength-resolved fluorescence and absorption study of the excited states of adenosine and an adenine oligomer[END_REF][START_REF] Borrego-Varillas | Exciton Trapping Dynamics in DNA Multimers[END_REF][START_REF] Karunakaran | Photoinduced dynamics of guanosine monophosphate in water from broad-band transient absorption spectroscopy and quantum-chemical calculations[END_REF][START_REF] Cheng | The solvent effect and identification of a weakly emissive state in nonradiative dynamics of guanine nucleosides and nucleotides -a combined femtosecond broadband time-resolved fluorescence and transient absorption study[END_REF] and considering the theoretical predictions for the excited state relaxation of dinucleotides with fully stacked chromophores, we examine below the experimental transient absorption spectra obtained for AG and GA. The spectral evolution is shown in Figure 3 and, not surprisingly, it is quite complex. The spectra recorded between 100 fs and 1 ps exhibit an intense UV band and a second broader and less intense one peaking in the 580 -600 nm region (Figures 3a and3b). During this time interval, the UV band shifts to shorter wavelengths. The same thing happens with the band in the visible, with the exception of an initial red shift between 100 and 200 fs. To better distinguish subsequent changes, which are not accompanied by an important modification of the signal intensity, the spectra obtained between 0.5 and 15 ps have been normalized at their maximum (Figures 3c and3d). In this time window, it appears that the UV band is composed in fact of two components. The high energy one, located below 350 nm, disappears within 15 ps. In parallel, the relative intensity of the band in the visible increases and eventually it becomes preponderant.

At longer times, no further spectral changes, within the experimental error, could be detected. We assign the blue shift of the bands in the UV and the visible occurring before 1 ps to the decay of the adenosine * state (La), [START_REF] Kwok | Femtosecond time-and wavelength-resolved fluorescence and absorption study of the excited states of adenosine and an adenine oligomer[END_REF][START_REF] Borrego-Varillas | Exciton Trapping Dynamics in DNA Multimers[END_REF][START_REF] Crespo-Hernàndez | Ultrafast excited state dynamics in nucleic acids[END_REF][START_REF] Improta | Quantum Mechanical Studies on the Photophysics and the Photochemistry of Nucleic Acids and Nucleobases[END_REF] whose photo-induced absorption spectrum peaks at longer wavelengths compared to that of guanosine (Figure 1c). The disappearance of the high energy component in the UV band, taking place within a few ps, is associated with the decay of the guanosine * state (Figure 1c). [START_REF] Karunakaran | Photoinduced dynamics of guanosine monophosphate in water from broad-band transient absorption spectroscopy and quantum-chemical calculations[END_REF][START_REF] Cheng | The solvent effect and identification of a weakly emissive state in nonradiative dynamics of guanine nucleosides and nucleotides -a combined femtosecond broadband time-resolved fluorescence and transient absorption study[END_REF] The small, albeit detectable, red shift of the band in the visible between 100 and 200 fs is tentatively attributed to exciton states associated with various Although the trends described in the previous paragraph are common for AG and GA, there are noticeable differences in both the spectral and the dynamical features of the two systems. Their spectra recorded at 100 fs, expected to correspond mainly to * states, look quite similar (Figure 3e). The largest difference is observed around 450 nm, where the relative intensity of the AG spectrum is higher. We are tempted to correlate this specific feature with the absorption of the exciton state identified only for AG, exhibiting a characteristic band in this spectral range (Figure 2a). However, as numerous transients enter into play, we cannot be conclusive about this assignment.

At 1 ps the high-energy peak is located at 353 nm for AG vs 349 nm for GA, in line with the relative position of the computed spectra for the excited states localized on the guanosine chromophore (Figures 2a and2b). The difference between the transient absorption spectra of the two dinucleotides increases with time.

Focusing on the dynamical aspects, the signal at 330 nm, associated mainly with the decay of the guanosine * state, is faster for AG than for GA (Figure 4a). This could be attributed to a faster formation of the CT state in the former system. This difference in the dynamics is also reflected in the spectra shown in Figures 3c and3d: the inversion in the relative intensity of the two bands takes place earlier for AG compared to GA.

We judge that, at short times, fits of the decays with multiexponential functions are inappropriate, due to the structural inhomogeneity of the studied systems and the anisotropic reactions taking place. In this respect, we recall that the decays of the free adenosine or guanosine in aqueous solution have been fitted with two or three exponentials each, the time constants depending on the probing wavelength and differing from one study to the other. 21, 30, 32- 33, 37 In any way, it is safe to consider that at 15 ps, all * states, both localized and excitonic, have disappeared and only the CT states are left. The signals recorded for AG and GA at selected wavelengths between 500 and 645 nm are well described by mono-exponential functions (Figures 4b and4c). The time constants derived from these fits fall, respectively, in the range of 112 ± 12 ps and 170 ± 10 ps. The former value is in good agreement with the lifetimes reported previously for AG exciplexes (105 ± 30 ps [START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF] and 124 ± 12 ps [START_REF] Balanikas | DNA Photodamage: From Light Absorption to Cellular Responses and Skin Cancer[END_REF] ) by transient absorption experiments probing in the UV/visible. A more recent study, performed for both AG and GA probing in the infrared spectral domain, provided much longer lifetimes: 420 ± 120 ps for AG and 280 ± 160 ps for GA. [START_REF] Kufner | UV-Induced Charge-Transfer States in Short Guanosine-Containing DNA Oligonucleotides[END_REF] As the error bars were quite large, the authors concluded that they could not discriminate between the two systems. From the above-described dynamical behavior, we conclude that the spectra at 15 ps, shown in Figure 3f after normalization at their UV peak, correspond to a single transient species, attributed to the CT state. Both spectra exhibit a UV band, peaking at 379 and 390 nm for AG and GA, respectively, and a broader and more intense one appearing at wavelengths longer than 500 nm.

The determination of the exact position of the latter band is hampered by a bandpass filter (300 -630nm) used to eliminate the scattered pump light. Despite this caveat, it is clear that the GA band is red-shifted in respect with that of AG. The relative position of both bands determined Although the present measurements do not allow the determination of absolute quantum yields associated with the formation of CT states in each dinucleotide, it is possible to estimate the relative quantum yields of their formation in the two systems. Considering that, under our experimental conditions, the same number of photons is absorbed by either system, the CT concentration in each system is determined by the intensity of the corresponding "zero-time" transient absorption spectrum. The latter are obtained from the transient absorption spectra at 15 ps and the lifetimes of the CT states, as explained in the SI. These "zero-time" spectra of the CT states are shown in Figure S3a. We remark that the intensity of the UV band, for which the experimental error is smaller compared to that of the band in the red, is ca. 75% higher for GA than for AG. This difference is unlikely to arise from larger values of the molar extinction coefficients. As a matter of fact, according to the computed spectra (Figure S3b), the maximum oscillator strength of the UV peak is 20% smaller for GA than for AG. Therefore, it appears the CT state is formed with a significantly higher quantum yield in the former system. The relative values of quantum yields can be explained, in part, by the more efficient coupling between CT and * states in the Franck-Condon region in GA, [START_REF] Balanikas | The Structural Duality of Nucleobases in Guanine Quadruplexes Controls Their Low-Energy Photoionization[END_REF] as well as by the presence of an additional relaxation pathway in AG leading to the exciton.

In conclusion, our joint experimental and theoretical study provided clear evidence about the different behavior of the excited state relaxation in the AG and GA dinucleotides, highlighting the influence of the DNA polarity on the CT states. The CT state in AG is formed faster and with a significantly lower quantum yield, absorbs at shorter wavelengths and decays faster compared to what is observed for its counterpart in GA. From a methodological point of view, this is the first time that computed spectra have been used to discriminate among transient species in DNA multimers determined by femtosecond spectroscopy.

It is worth-noticing that our results obtained upon DNA excitation are in line with those observed for redox reactions between ground state DNA and an external oxidant: they are all favored when guanines are located at 5' ends. [START_REF] Saito | Photoinduced DNA Cleavage via Electron Transfer -Demonstration that Guanine Residues located 5' to Guanine are the Most Electron-donating sites[END_REF][START_REF] O'neill | Effects of strand and directional asymmetry on base-base coupling and charge transfer in double-helical DNA[END_REF] Moreover, the catalytic activity of G4-DNAzymes increases with the availability of guanines at 5' ends. [START_REF] Virgilio | Improved performances of catalytic G-quadruplexes (G4-DNAzymes) via the chemical modifications of the DNA backbone to provide G-quadruplexes with double 3 '-external G-quartets[END_REF][START_REF] Cao | Manipulating the Assembly of DNA Nanostructures and Their Enzymatic Properties by Incorporating a 5 '-5 ' Polarity of Inversion Site in the G-Tract[END_REF] Finally, the present study shed light on the multistep mechanism underlying low-energy photoionization of DNA, proposed to involve formation of CT states, charge separation and electron ejection by the nucleobase bearing the negative charge. [START_REF] Balanikas | Guanine Radicals Induced in DNA by Low-Energy Photoionization[END_REF][START_REF] Balanikas | DNA Photodamage: From Light Absorption to Cellular Responses and Skin Cancer[END_REF] The ca. 30% higher photoionization quantum yield determined at 266 nm for guanine quadruplexes composed of four DNA strands 5'-GGGGA-3' (12.6×10 -3 ) compared to 5'-AGGGG-3' strands (9.9 ×10 -3 ), [START_REF] Balanikas | The Structural Duality of Nucleobases in Guanine Quadruplexes Controls Their Low-Energy Photoionization[END_REF] can be explained by the formation of higher concentration of G +  A -CT states in the former system, accompanied by a longer lifetime, favoring charge separation and trapping of the electron hole by the stacked guanines. [START_REF] Saito | Mapping of the hot spots for DNA damage by one-electron oxidation: Efficacy of GG doublets and GGG triplets as a trap in long-range hole migration[END_REF] Supporting Information. Sample details; transient absorption setups; relative quantum yields of CT states; computational details.

Figure 1 .

 1 Figure 1. (a) Schematic representation of the geometrical arrangement adopted by the adenine (red) and guanine (green) moieties in the ground state of AG and GA. (b) Steady-state absorption spectra of AG (blue), GA (red) and an equimolar mixture of dAMP and dGMP (black). 31 The vertical violet line indicates the excitation wavelength used for the transient absorption experiments, 266 nm, at which the spectra have been normalized.

29

 29

Figure 2 .

 2 Figure 2. Transient absorption spectra computed for the minima identified in the potential energy surface of the first excited state of AG (a) and GA (b); green: * state localized on G; cyan: exciton state delocalized over A and G; blue: CT state in AG; red: CT state in GA; the spectra of the CT states are compared in (c) after normalization of their intensity at the UV peak.

Figure 3 .

 3 Figure 3. Transient absorption spectra recorded for AG (a and c) and GA (b and d); the spectra in (a) and (b) are recorded from 100 fs to 1 ps with 100 fs steps; the spectra in (c) and (d) are normalized in their maximum. The spectra of AG (blue) and GA (red) at 100 fs and 15 ps, normalized at the UV peak, are compared in (e) and (f), respectively.

  general, they are expected to undergo fast localization in such flexible systems.

Figure 4 .

 4 Figure 4. Decays recorded for AG (blue) and GA (red) at 330 nm (a) and 500 nm (b) and (c). The signals in (a) are normalized at their maximum and those in (b) and (c) at 15 ps. Yellow lines correspond to fits with mono-exponential functions.

  two dinucleotides is reminiscent of what is observed for the computed spectra of their CT states (Figure2c): both bands of AG are located at shorter wavelengths compared to those of GA.

ACKNOWLEDGMENT

This work received funding by the European Program H2020 MSCA ITN (grant No. 765266 -LightDyNAmics project)