Isabelle Testard

Elizabeth Garcia-Chartier

Amani Issa

Véronique Collin-Faure

Catherine Aude-Garcia

Serge M Candéias
email: serge.candeias@cea.fr

Bystander signals from low-and high-dose irradiated human primary fibroblasts and keratinocytes modulate the inflammatory response of peripheral blood mononuclear cells

Keywords: bystander effects (BEs), inflammation, low-dose radiation, high-dose radiation, gene expression, cytokines •

Irradiated cells can propagate signals to neighboring cells. Manifestations of these so-called bystander effects (BEs) are thought to be relatively more important after exposure to low-vs high-dose radiation and can be mediated via the release of secreted molecules, including inflammatory cytokines, from irradiated cells. Thus, BEs can potentially modify the inflammatory environment of irradiated cells. To determine whether these modifications could affect the functionality of bystander immune cells and their inflammatory response, we analyzed and compared the in vitro response of primary human fibroblasts and keratinocytes to low and high doses of radiation and assessed their ability to modulate the inflammatory activation of peripheral blood mononuclear cells (PBMCs). Only high-dose exposure resulted in either up-or down-regulation of selected inflammatory genes. In conditioned culture media transfer experiments, radiation-induced bystander signals elicited from irradiated fibroblasts and keratinocytes were found to modulate the transcription of inflammatory mediator genes in resting PBMCs, and after activation of PBMCs stimulated with lipopolysaccharide (LPS), a strong inflammatory agent. Radiation-induced BEs induced from skin cells can therefore act as a modifier of the inflammatory response of bystander immune cells and affect their functionality.

INTRODUCTION

DNA lesions and activation of DNA damage signaling pathways can readily be observed in cells after irradiation [START_REF] Huang | DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer[END_REF][START_REF] Lomax | Biological consequences of radiation-induced DNA damage: relevance to radiotherapy[END_REF]. More surprising, DNA lesions, or their consequences, can also occur in cells that have not been irradiated but are located in the vicinity of exposed cells [START_REF] Nagasawa | Induction of sister chromatid exchanges by extremely low doses of alpha-particles[END_REF]. This phenomenon has been termed non-targeted effects (NTEs) or bystander effects (BEs). BEs have been demonstrated in many different experimental systems. They can be transmitted by direct cell-cell contact or via factors released in culture medium [START_REF] Morgan | Non-targeted effects induced by ionizing radiation: mechanisms and potential impact on radiation induced health effects[END_REF][START_REF] Prise | Radiation-induced bystander signalling in cancer therapy[END_REF][START_REF] Wright | Manifestations and mechanisms of non-targeted effects of ionizing radiation[END_REF] and propagated through recipient cells [START_REF] Al-Mayah | The non-targeted effects of radiation are perpetuated by exosomes[END_REF][START_REF] Fu | Macrophage-mediated bystander effects after different irradiations through a p53-dependent pathway[END_REF] and in tissues [START_REF] Belyakov | Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away[END_REF], possibly by microvesicles/exosomes [START_REF] Al-Mayah | Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation[END_REF][START_REF] Ariyoshi | Radiation-induced bystander effect is mediated by mitochondrial DNA in exosome-like vesicles[END_REF][START_REF] Moertl | Radiation exposure of peripheral mononuclear blood cells alters the composition and function of secreted extracellular vesicles[END_REF][START_REF] Szatmari | Extracellular vesicles mediate low dose ionizing radiation-induced immune and inflammatory responses in the blood[END_REF]. Bystander cells can be irradiated or not. BEs can therefore act as a modifier of the response to radiation of irradiated cells [START_REF] Blyth | Radiation-induced bystander effects: what are they, and how relevant are they to human radiation exposures?[END_REF][START_REF] Cary | Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation[END_REF].

As NTEs were first showed to replicate in non-exposed cells some of the effects induced by radiation in irradiated cells, most of the endpoints analyzed were associated by the induction of-or the response to-genotoxic stress in bystander cells [START_REF] Nagasawa | Induction of sister chromatid exchanges by extremely low doses of alpha-particles[END_REF][START_REF] Fu | Macrophage-mediated bystander effects after different irradiations through a p53-dependent pathway[END_REF][START_REF] Belyakov | Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away[END_REF][START_REF] Cary | Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation[END_REF][START_REF] Xiao | Irradiated U937 cells trigger inflammatory bystander responses in human umbilical vein endothelial cells through the p38 pathway[END_REF][START_REF] Hargitai | Oxidative stress and gene expression modifications mediated by extracellular vesicles: an in vivo study of the radiation-induced bystander effect[END_REF][START_REF] Portess | Low-dose irradiation of nontransformed cells stimulates the selective removal of precancerous cells via intercellular induction of apoptosis[END_REF][START_REF] Sokolov | Global gene expression alterations as a crucial constituent of human cell response to low doses of ionizing radiation exposure[END_REF][START_REF] Lepleux | Bystander effectors of chondrosarcoma cells irradiated at different LET impair proliferation of chondrocytes[END_REF]. Several pathways have been involved in the transmission of these effects, including MAPK [START_REF] Cary | Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation[END_REF][START_REF] Xiao | Irradiated U937 cells trigger inflammatory bystander responses in human umbilical vein endothelial cells through the p38 pathway[END_REF], p53 [START_REF] Fu | Macrophage-mediated bystander effects after different irradiations through a p53-dependent pathway[END_REF], NF-kB [START_REF] Zhou | Mitochondrial function and nuclear factor-kappaB-mediated signaling in radiation-induced bystander effects[END_REF], cytokine/cytokine receptor interactions [START_REF] Lorimore | Chromosomal instability in unirradiated hemaopoietic cells induced by macrophages exposed in vivo to ionizing radiation[END_REF][START_REF] Shao | Role of TGF-β1 and nitric oxide in the bystander response of irradiated glioma cells[END_REF], and modulation of the redox status [START_REF] Hargitai | Oxidative stress and gene expression modifications mediated by extracellular vesicles: an in vivo study of the radiation-induced bystander effect[END_REF][START_REF] Portess | Low-dose irradiation of nontransformed cells stimulates the selective removal of precancerous cells via intercellular induction of apoptosis[END_REF][START_REF] Lorimore | Chromosomal instability in unirradiated hemaopoietic cells induced by macrophages exposed in vivo to ionizing radiation[END_REF][START_REF] Shao | Role of TGF-β1 and nitric oxide in the bystander response of irradiated glioma cells[END_REF][START_REF] Lyng | Reactive oxygen speciesinduced release of signalling factors in irradiated cells triggers membrane signalling and calcium influx in bystander cells[END_REF]. BEs involve a large number of different proteins in irradiated cells, conditioned medium or bystander cells [START_REF] Chevalier | Proteomic overview and perspectives of the radiation-induced bystander effects[END_REF][START_REF] Zhang | High and low doses of ionizing radiation induce different secretome profiles in a human skin model[END_REF][START_REF] Yentrapalli | Quantitative changes in the protein and miRNA cargo of plasma exosomelike vesicles after exposure to ionizing radiation[END_REF][START_REF] Kis | The effect of ionising radiation on the phenotype of bone marrow-derived extracellular vesicles[END_REF]. Interestingly, BEs have been suggested to saturate above a certain radiation threshold [START_REF] Nagasawa | Induction of sister chromatid exchanges by extremely low doses of alpha-particles[END_REF][START_REF] Portess | Low-dose irradiation of nontransformed cells stimulates the selective removal of precancerous cells via intercellular induction of apoptosis[END_REF][START_REF] Belyakov | Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts[END_REF]; consequently, their contribution to the overall radiationinduced effects might be relatively more important after low-dose radiation exposure [START_REF] Morgan | Non-targeted effects induced by ionizing radiation: mechanisms and potential impact on radiation induced health effects[END_REF][START_REF] Prise | Radiation-induced bystander signalling in cancer therapy[END_REF]. However, supernatant transfer experiments did show that BEs can be elicited from cells exposed to high-dose radiation [START_REF] Cary | Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation[END_REF][START_REF] Xiao | Irradiated U937 cells trigger inflammatory bystander responses in human umbilical vein endothelial cells through the p38 pathway[END_REF][START_REF] Muller | Radiation-induced mast cell mediators differentially modulate chemokine release from dermal fibroblasts[END_REF][START_REF] Rzeszowska-Wolny | X-irradiation and bystander effects induce similar changes of transcript profiles in most functional pathways in human melanoma cells[END_REF].

Inflammation is a defense mechanism resulting in the mobilization of immune cells in response to infection or tissue damage. The sensing of pathogens or tissue damage involves some of the same receptors on immune cells, including toll-like receptors (TLRs). The so-called sterile inflammation is initiated by molecules released/produced by tissue damage, stressed cells or cells dying by non-apoptotic mechanisms, collectively termed danger signals or danger associated molecular patterns (DAMPs) [START_REF] Candeias | The many interactions between the innate immune system and the response to radiation[END_REF][START_REF] Chen | Sterile inflammation: sensing and reacting to damage[END_REF]. These molecules include ATP, chromatin/DNA, cytokines and intra-cellular molecules like HMGB1 or uric acid [START_REF] Chen | Sterile inflammation: sensing and reacting to damage[END_REF][START_REF] Rider | Alarmins: feel the stress[END_REF]. Radiation-induced sterile inflammation can be seen as a category of bystander signalization whereby irradiated cells alert immune cells that they have been damaged. [START_REF] Szatmari | Extracellular vesicles mediate low dose ionizing radiation-induced immune and inflammatory responses in the blood[END_REF] In vivo, the sensing of extra-cellular danger signals by TLRs can modulate the activity of immune cells. For example, radiation exposure can lead to long lasting activation of inflammatory networks, including some usually activated after DAMPs recognition by TLRs and potentiation of TLR signaling [START_REF] Tajima | Immune system phenotyping of radiation and radiation combined injury in outbred mice[END_REF]. Interestingly, these TLR-dependent events are strongly reduced when the production of uric acid/uric crystal is inhibited in mice [START_REF] Stoecklein | Radiation exposure induces inflammasome pathway activation in immune cells[END_REF]. In vitro, the role of immune cells as donor [START_REF] Moertl | Radiation exposure of peripheral mononuclear blood cells alters the composition and function of secreted extracellular vesicles[END_REF][START_REF] Xiao | Irradiated U937 cells trigger inflammatory bystander responses in human umbilical vein endothelial cells through the p38 pathway[END_REF], receiver [START_REF] Cary | Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation[END_REF], or transmitter [START_REF] Fu | Macrophage-mediated bystander effects after different irradiations through a p53-dependent pathway[END_REF] of bystander signals have been addressed in different studies using freshly purified blood peripheral mononuclear cells (PBMCs) [START_REF] Fu | Macrophage-mediated bystander effects after different irradiations through a p53-dependent pathway[END_REF][START_REF] Moertl | Radiation exposure of peripheral mononuclear blood cells alters the composition and function of secreted extracellular vesicles[END_REF] or immortalized human cell lines [START_REF] Fu | Macrophage-mediated bystander effects after different irradiations through a p53-dependent pathway[END_REF][START_REF] Cary | Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation[END_REF][START_REF] Xiao | Irradiated U937 cells trigger inflammatory bystander responses in human umbilical vein endothelial cells through the p38 pathway[END_REF]. These studies mostly assessed the induction of genotoxic effects in receiver cells exposed to bystander signaling elicited by high-dose exposure but did not investigate whether and how these bystander signals modify the ability of immune cells to be activated and respond to an inflammatory stimulus.

In this article, we compared the response of primary fibroblasts and keratinocytes from the same donors to low-and high-dose radiation exposure, and analyzed the impact of radiation-induced bystander signals elicited from these cells on the inflammatory status and response of peripheral blood mononuclear cells. Fibroblasts and keratinocytes were found to produce distinct profiles of secreted cytokines, which are not modulated after exposure to low-or high-dose radiation. However, conditioned culture media from these cells modulate PBMCs inflammatory status and activation, with slight differences according to the cell type and exposure. We concluded that radiation-induced effects have the ability to modify the functionality of immune cells, and these effects appear to be distinct for each cell lineage.

MATERIALS AND METHODS

Cell culture and reagents

Primary skin cells were a generous gift from M Martin's laboratory (CEA, Evry, France). Primary skin keratinocytes (human primary keratinocytes [HKPM]) and fibroblasts (human primary fibroblasts [HFPM]) have been isolated from biopsies from three healthy female donors, numbered 168, 170 and 172, after their informed consent, as described in [START_REF] Alexaline | Bioengineering a human plasma-based epidermal substitute with efficient grafting capacity and high content in clonogenic cells[END_REF][START_REF] Fortunel | Exploration of the functional hierarchy of the basal layer of human epidermis at the singlecell level using parallel clonal microcultures of keratinocytes[END_REF]. Cells were amplified in order to constitute banks of each cell types and donors in passage 1-2. For routine culture, and after division, fibroblasts and keratinocytes were seeded in 75 cm 2 culture flasks at a density of 5000 and 800 cells/cm 2 respectively. Three to four replicates were used in all experiments for each cell type.

Fibroblasts were cultured in DMEM (Gibco ® DMEM + Gluta-MAXTM, Fischer Scientific, Illkirch, France) supplemented with 10% fetal bovine serum (FBS, Eurobio Scientific, Les Ullis, France) and 1% of penicillin-streptomycin antibiotics (Gibco ® , Pen Strep, Fischer Scientific, Illkirch, France). For keratinocytes, we used a semi-defined KBM-2 culture medium (Lonza Clonetics ® , KBM-2 keratinocyte basal medium-2, supplemented with KGM-2 Singlequots, Ozyme, St Cyr l'Ecole, France). The cells were grown at 37 • C in a humidified incubator with 5% CO 2 . Medium was changed every 2 days until the cells reached 70-80% confluence. Then fibroblasts were passed in a medium containing only 0.1% FBS for 5 days before their irradiation in order to maintain most cells in a quiescent state. Nutlin-3 and lipopolysaccharide (LPS) from E. coli 0111:B4 were purchased from Sigma Aldrich. Nutlin-3 stock solution was prepared and diluted in dimethylsulfoxyde (DMSO). It was added for the indicated time to cells prepared as described above. Untreated cells received the same volume of DMSO.

Irradiation

Keratinocytes were at passage 2 or 3 and fibroblasts between passage 4 and 7 at the time of radiation exposure in T75 culture flasks. Their respective culture medium was renewed 24 hours before irradiation. Duplicate flasks were exposed at room temperature to low (50, 100 mGy), and high (2 Gy and 10 Gy) doses of 60 Co γ -rays, using the Anémone Bio irradiator of ARC-Nucléart facility (CEA-Grenoble). Dose rates were 0.034 Gy / min for low doses, and 0.524 Gy / min for high doses. Control cells (non-irradiated cells) were subjected to the same conditions as irradiated cells (sham irradiation). After irradiation, cells were incubated at 37 • C in a humidified incubator with 5% CO 2 , for 24 h and 48 h post exposure. In these conditions, cell mortality, measured by Trypan blue exclusion or by using an automated Scepter™ Cell Counter (Merck), never exceeded 20% after 10 Gy exposure.

Supernatants and cells collection

Supernatants from cells irradiated at the different doses were collected 24 and 48 hours after exposure to γ -rays. They were centrifuged (10 min at 1400 rpm) and filtered (0.22 μM) in order to discard cells fragments, and aliquots were then frozen at -20 • C until use.

After the collection of supernatants, the cells were harvested by trypsinization after two washes in PBS. Trypsin-EDTA (Fischer Scientific, Illkirch, France) was used at 0.25% for fibroblasts or diluted to 0.05% for keratinocytes for 5 minutes at 37 • C, then inactivated with FBS (20% final) and finally rinsed twice in D-PBS.

RNA purification-quantitative real-time PCR analysis

Total RNA was extracted from cells at the indicated times with the Nucleospin RNA/Protein purification kit (Macherey-Nagel, Hoerdt, France), following the manufacturer's protocol. RNA was eluted in RNAse-free water and quantified using a NanoDrop-2000 spectrophotometer.

Total RNA (0.6 to 1 μg) was converted into cDNA by priming with oligo-dT using the HS RT-PCR kit (Sigma-Aldrich France, L'lsle D'Abeau Chesnes, France) according to manufacturer's protocol and diluted to a final volume of 50 μL. A control reaction without reverse transcriptase (noRT control) was included in each series of samples.

Real-time PCR was performed in 384 well plates, in a C1000 TM thermal cycler (BioRad Life Sciences, Marnes-la-Coquette, France), in a final volume of 10 μL containing 5 μL of Lumino CT reaction mix (Sigma-Aldrich France, L'lsle D'Abeau Chesnes, France), 2 μL of cDNA diluted 1/8 and 2 μL of primers at 2 mM each. After 20 s of denaturation at 95 • C, amplification was performed for 40 cycles consisting of 5 s of denaturation at 95 • C and 20 s of elongation at 60 • C. All reactions were carried out in triplicate. The specificity of the reactions was verified by a melting curve analysis of the products generated. Amplification of the noRT control sample was used to check the absence of contaminating genomic DNA in our RNA preparations. Data were collected and analyzed with CFX Manager 3.1 software (BioRad Life Sciences, Marnes-la-Coquette, France). We used primers designed to amplify p53-dependent stress response genes (CCNG1, DDB2, PHPT1 and CDKN1A, coding for the p21 protein) and genes coding for inflammatory mediators (CSF1, CXCL8/IL-8, IL1B, MCP-1, TGFB1 and TNFA). PCR reactions were normalized using two housekeeping genes (HPRT and 36B4). The sequence of the primers is provided in Supplementary Table S1. PCR runs were validated when the amplification of the housekeeping genes resulted in a coefficient of variation and M-values < 0.25 and < 0.5, respectively, as calculated by the CFX manager software, indicating that they are not differentially regulated in our experimental conditions.

Quantification of cytokines secretion

We simultaneously measured the secretion of IL-6, IL-8, MIP1α, GM-CSF, MCP-1 and TNFα in supernatants of control and irradiated HFPM and HKPM cells collected 24 and 48 hours post-exposure using multiplex ELISA on bead assays (Cytometric Bead Arrays, Becton-Dickinson Life Sciences-Biosciences, Le Pont-de-Claix, France) according to manufacturer's instruction. Results were analyzed using the FCAP array software (Becton-Dickinson Life Sciences-Biosciences, Le Pont-de-Claix, France), and normalized on the number of live cells in each condition.

Functional analysis of BEs

PBMCs were purified from buffy coats purchased from the French Blood Bank (Etablissement Français du Sang -Rhone-Alpes, La Tronche, France, under contract #15-2041) by density gradient using UNI-SEP tubes (NOVAmed, Eurobio Scientific, Les Ulis, France) according to manufacturer's instructions. These PBMCs were then resuspended at a concentration of 2 to 4 × 10 6 cells/mL in supernatants from control and irradiated HFPM and HKPM cells, and incubated at 37 • C in a humidified incubator with 5% CO2 without or with LPS (1 ng/mL) for 20 h in 6-wells plates.

RNA prepared from PBMCs cultured in HFPM168 and HKPM168 supernatants was used to quantify the expression of cytokine (IL1B, CCL3, CXCL8/IL8) and stress response (DDB2, PHPT1, CCNG1) genes as described above. PCR results from 3 replicate experiments were analyzed with the CFX Manager 3.1 software 'volcano plot' option to compare the modulation of gene expression in PBMCs incubated in irradiated vs control HFPM and HKPM conditioned culture media, with a fold change threshold of 1.6 (arbitrarily selected) to identify genes up-or down-regulated with a P-value < 0.05 by a t-test.

Culture supernatants were harvested as above to quantify the secretion of IL-1β, IL-6, IL-8 and MCP-1 by PBMCs by FACS, using multiplex ELISA on beads assays as described above. Results were analyzed using the FCAP array software (Becton-Dickinson) and expressed normalized on cytokine secretion by PBMCs cultured in the supernatant of non-irradiated fibroblasts and keratinocytes.

Statistical analysis

Statistical analysis was performed using the Past 4.03 freeware [START_REF] Hammer | PAST: paleontological statistics software package for education and data analysis[END_REF]. Multiple comparisons were performed by an ANOVA test followed by Tuckey's pairwise comparison test if the data were normally distributed, or a Kruskal-Wallis test followed by a Dunn's post hoc test is the distribution was not normal. Normality was determined by the Shapiro-Wilk test. For pairwise comparisons, a t-test was performed.

RESULTS

Experimental strategy

The experimental strategy devised to address the potential modifier role of radiation-induced bystander signals produced from irradiated primary fibroblasts and keratinocytes onto the inflammatory response is depicted in Fig. 1. Primary fibroblasts and keratinocytes were exposed to γ -ray doses ranging from 0.05 to 10 Gy. These cells and their culture media were harvested 24 h and 48 h after exposition to analyze the direct effects of radiation exposure, more specifically on the expression of a panel of genes coding for inflammatory factors. This panel includes genes coding for chemokines (CSF1, CXCL8/IL-8, MCP-1) and pro-(IL1B, TNFA) and anti (TGFB1)-inflammatory cytokines to analyze the eventual regulation of a broad spectrum of potential activities on immune cells. In addition, the conditioned culture media harvested from un-irradiated control and irradiated fibroblasts and keratinocytes was used to cultivate purified PBMCs for 24 h, either in a resting state (no LPS) or activated by LPS (+LPS). We then measured the transcription of stress-response and inflammatory genes in these PBMCs, and their secretion of inflammatory cytokines to analyze the indirect effects of the exposure of fibroblasts and keratinocytes.

Modulation of stress and inflammatory responses in primary fibroblasts exposed to low-and high-dose radiation

HFPM from three different donors (donors 168, 170 and 172) were exposed to low (50 mGy, 100 mGy) and high (2 Gy, 10 Gy) doses of γ -radiation from a 60 Co source. Cells and their supernatants were harvested 24 and 48 hours later as described in Materials and Methods. We compared in these cells the relative expression of a panel of stress-responsive, p53-dependent genes (CCNG1, DDB2, CDKN1A, PHPT1) and of a panel of genes coding for inflammatory cytokines (CSF1, CXCL8/IL-8, MCP-1, TGFB1, IL1B, TNFA) by RT-qPCR 24 h and 48 h after exposure (Fig. 2). Low-dose exposure did not affect the relative expression any of these genes when compared to un-irradiated control cells. High-dose exposure induced a significant up regulation of the stress response genes 24 h post irradiation when compared to control or low-dose exposed cells, which was maintained or slightly increased at 48 h. The increase in gene expression is radiation-dose dependent for DDB2 and CDKN1A at 24 h, but only for CDKN1A at 48 h. The modulation of cytokine gene expression after high-dose exposure is clearly different. The relative expression of IL-8 is downregulated in cells exposed to 10 Gy when compared to un-irradiated and 2 Gy exposed cells at 24 h and 48 h, respectively. The relative expression of MCP-1 is significantly increased only after exposure to 2 Gy and always higher than in 10 Gy exposed cells, at both 24 h and 48 h. Transcription of TGFB1 is increased after 10 Gy at both time, but after 2 Gy only at 48 h when compared to un-irradiated control cells and cells exposed to low-dose radiation. The relative expression of IL1B and TNFA was not reliably detected in these cells. Thus, in primary fibroblasts, we do not observe any transcriptional regulation of this panel of inflammatory genes after low-dose exposure, and exposure to high doses induces can result in sporadic increase or decrease of specific genes.

To find out whether the transcription of cytokine genes can be induced by a more sustained activation of the DNA damage response (DDR) mechanisms activated after radiation exposure, primary fibroblasts were treated with nutlin-3, which activates the p53 pathway by inhibiting p53-MDM2 interactions [START_REF] Vassilev | In vivo activation of the p53 pathway by small-molecule antagonists of MDM2[END_REF]. In HFPM168 cells treated with nutlin-3 (1 and 10 μM) for 6 or 24 h, we observed, as expected, a clear up-regulation of the transcription of all the stress response genes, but also of most of the inflammatory cytokine genes analyzed (Supplementary Fig. S1). A dose-dependent induction of CCNG1 and CDKN1A expression is already evident after 6 h of stimulation, and is maintained until 24 h, whereas DDB2 and PHPT1 induction become nutlin-3 dose-dependent only at that time. In contrast, transcriptional up-regulation of cytokine genes occurs only in cells treated with 10 μM nutlin-3. This increase already occurs after 6 h of culture for CSF1 and IL1B, but only after 24 h for CXCL8/IL-8 and MCP-1. The transcription of TGFB1 is not significantly affected by p53 activation. Thus, in primary fibroblasts, a strong and sustained pharmacological activation of p53 can markedly induce the transcription of inflammatory mediators genes, but exposure to low-and high-dose radiation have at most a very limited effect.

RT-PCR analysis only offers a snapshot of gene expression at the time the cells were harvested. Indeed, cytokine production can also be regulated at the post-transcriptional level and/or at the level of secretion [START_REF] Murray | Cytokine secretion in macrophages: SNAREs, Rabs, and membrane trafficking[END_REF]. Thus, to find out whether cytokine secretion was regulated after radiation exposure, we measured the accumulation of a series of cytokines (IL-6, IL-8, MIP1α, GM-CSF, MCP-1 and TNFα) in the conditioned culture medium of HFPM168 and HFPM170 cells exposed to low (50 and 100 mGy) and high (2 Gy) doses of radiation, 24 h and 48 post exposure. We did not consider supernatants from HFPM cells exposed to 10 Gy in these analyses to avoid an eventual interference of cytokines released rather than secreted after cell death, even though viability was always greater than 80% after 10 Gy exposure. Only IL-6, IL-8 and MCP-1 could be reliably detected. HFPM168 and HFPM170 cells secreted similar levels of IL-8 and MCP-1, but the former was found to produce more IL-6 than the latter (Supplementary Fig. S2). The secretory profiles of these three cytokines is not affected by radiation and time post exposure (Fig. 3, left) We concluded that exposure to low-or high-dose radiation does not significantly modulate the secretion of these cytokines in the 2 days following exposure.

Modulation of stress and inflammatory responses in primary keratinocytes exposed to low-and high-dose radiation

The same experiments were performed on HKPM obtained from the same donors (donors 168, 170 and 172), with the difference that only HKPM170 cells were exposed to 10 Gy. The effects of radiation on primary keratinocytes were essentially the same than in primary fibroblasts 24 h after high-dose exposure, even though the level of induction was lower. In addition, in contrast to the results obtained with fibroblasts, the up-regulation of stress genes expression is not maintained at day 2 post exposure. The relative expression of DDB2, CDKN1A and PHPT1 after 2 Gy exposure is for example no longer significantly higher than that of cells exposed to low-dose radiation at 48 h (Fig. 4). Unlike in fibroblasts, we could not reliably detect CSF1 transcription in keratinocytes while IL1B and TNFA genes were readily amplified. Again, radiation exposure had limited effects on cytokine gene expression. IL1B and TGFB1 expression was slightly up-regulated 24 h after 2 Gy, and that of TGFB1 after 2 and 10 Gy exposure. The relative expression of CXCL8/IL-8 and TNFA was decreased in cells exposed to high-dose radiation. These modifications were only transient as these changes were no longer visible 48 h post exposure. Thus, here again, we do not observe any modulation of the relative expression of inflammatory genes after low-dose exposure. Exposure to high doses results in up-or down-regulation of the transcription of specific genes, but, in contrast to primary fibroblasts, these changes are transient. The expression of these genes was then analyzed in HKPM168 cells treated by low (1 μM) and high (10 μM) doses of nutlin-3 for 6 or 24 h (Supplementary Fig. S1). In contrast to the results obtained with primary fibroblasts, the expression of the stress-response genes is significantly increased only in cells treated with 10 μM nutlin-3. Furthermore, the expression of DDB2 and PHPT1 is induced only after 24 h of stimulation. A similar profile is observed for the relative expression of IL1B and TGFB1, while that of the IL-8 gene is not significantly modified in any condition, when compared to untreated cells. Thus, nutlin-3 appears to be less potent on primary keratinocytes than on primary fibroblasts. These results nonetheless show that prolonged activation of p53, but not radiation exposure, can induce the transcription of at least some of the inflammatory cytokine genes.

Finally, as for fibroblasts, we measured the accumulation of IL-6, IL-8, MIP1α, GM-CSF, MCP-1 and TNFα in the medium of HKPM168 and HKPM170 cells 24 h and 48 h after exposure to low (50 and 100 mGy) and high (2 Gy) dose radiation. HKPM168 and HKPM 170 cells were found to produce similar levels of IL-8 (Supplementary Fig. S2), whereas HKPM170 produced in Fig. 3. Effects of radiation exposure on inflammatory cytokine secretion by primary fibroblasts and keratinocytes. The level of IL-6, IL-8, MIP1α, GM-CSF, TNFα and MCP-1 was measured in supernatants of HFPM (left) and HKPM (right) cells one and two days after exposure to the doses of radiation indicated on the x axis (Gy). Results for fibroblasts were obtained from HFPM168 (n = 3) and HFPM170 (n = 3). Results from keratinocytes were obtained from HKPM168 (n = 3) and HKPM170 (n = 4). HKPM168 was found to secrete only IL-8; MIP1α and TNFα were not detected in any condition. For each cytokine, the level of secretion in irradiated cells was compared to control cells by an ANOVA test. No significant differences were found (P > 0.05). The graphs represent mean ± SEM. Fig. 4. Modulation of stress and inflammatory gene expression in irradiated primary keratinocytes. The expression of stress (left) and inflammatory (right) genes was analyzed by RT-qPCR 24 h (top) and 48 h (bottom) after exposure of HKPM168 (n = 4), HKPM170 (n = 4) and HKPM172 (n = 4) cells to 0.05, 0.1, 2 and 10 Gy of γ -radiation. Only HKPM170 and 172 cells were found to transcribe the TNFA gene at a detectable level. The x axis (Gy) indicates the dose of radiation. Only HKPM170 were exposed to 10 Gy. For each gene, the level of expression in control and exposed cells was compared by an ANOVA test if the data were normally distributed or by a Kruskal-Wallis test, if they were not. When a value of P < 0.05 indicated significant differences in irradiated cells, pairwise comparisons were performed with a Tukey's test, after an ANOVA test, or a Dunn's post hoc test, after a Kruskal-Wallis test. The graphs represent mean ± SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001. addition IL-6 and GM-CSF (Fig. 3, right). None of the apparent changes observed with radiation and time post exposure are significant.

Influence of BEs on the inflammatory response of PBMCs

Even though we did not observe any significant changes in cytokine secretion by low-and high-dose irradiated primary fibroblasts and keratinocytes, we wanted to determine whether radiation-induced changes could have functional consequences and modulate the inflammatory status and/or response of bystander immune cells.

To this end, we cultured freshly purified PBMCs for 24 h in conditioned culture media harvested from control and irradiated keratinocytes and fibroblasts 24 h and 48 h after exposure to 0.05, 0.1 or 2 Gy, in absence of additional stimulation or with LPS. LPS exposure induces an inflammatory reaction and results in a strong up-regulation of cytokine production by activated PBMCs (Supplementary Fig. S3). The BEs induced by the conditioned media were assessed at the transcriptional level, by the quantification of cytokine and stress response genes expression, and by measuring the modulation of LPS-induced cytokine secretion by PBMCs. This experimental setting allows us to investigate whether and how bystander signaling affects PBMCs inflammatory status and response, i.e. their functionality.

HFPM168 supernatants harvested 24 h post 0.05 and 0.1 Gy exposure specifically reduced by 40% or more (FC < -1.6, P < 0.05) the transcription of IL1B (P = 0.054 in 0.05 Gy supernatants), CCL3 and CXCL8/IL8 genes in PBMCs when compared to nonirradiated HFPM168 (Fig. 5). Supernatants harvested from 2 Gy exposed fibroblasts only induced a reduction of CCL3 gene expression (P = 0.051). In 48 h HFPM168 supernatants the level of IL1B, CCL3 and CXCL8/IL8 (FC = -1.59) were coordinately down regulated in PBMCs only when fibroblasts were exposed to 0.05 Gy; IL1B expression was also found reduced (FC = -1.59) after 2 Gy exposure when compared to non-irradiated control supernatants. The transcription of DDB2, CCNG1 and PHPT1 was not modulated in PBMCs in any of these conditions. The reduction in cytokine gene expression in irradiated HFPM168 supernatants is lost when PBMCs are stimulated with LPS (data not shown). Conditioned culture media collected from control and irradiated HKPM168 cells had no effects of inflammatory and stress response gene expression in PBMCs, in absence or in presence of LPS (Fig. 5 and data not shown). Thus, signals resulting from irradiated fibroblasts, but not keratinocytes of a same donor are able to specifically modulate the transcription of inflammatory genes in bystander immune cells.

Next, to find out whether these effects on the inflammatory status of PMBCs translate at the level of their inflammatory response, we measured their secretion of cytokines as resting PMBCs and after LPS activation. In conditioned media obtained from control and irradiated fibroblasts (HFPM168 and HFPM170), resting PBMCs produced only IL-6 and MCP-1, but at levels that did not differ from the levels produced by un-irradiated primary fibroblasts. We therefore could not conclude on the origin of these cytokines. However, their levels were not modulated in supernatants from irradiated fibroblasts (Supplementary Fig. S4). LPS-stimulated PBMCs produced MCP-1, IL-6 and IL-1β. IL-1β secretion was decreased in conditioned media collected from low-and high-dose exposed HFPM cells 24 h post irradiation when compared to non-irradiated control supernatants (Fig. 6). This modulation appears to evolve with time and/or exposure as these effects persisted only in 48 h high-dose supernatants. LPSinduced production of MCP-1 and IL-6 is not significantly affected in the same conditions, suggesting that the reduced IL-1β secretion does not result from PBMCs death.

Resting PBMCs cultured in conditioned culture media harvested from un-irradiated primary keratinocytes produced IL-6, IL-8 and MCP-1. Here again we could not ascertain the origin of IL-6 and IL-8, as the levels measured were similar to that found in keratinocyte culture media. However, similar to the situation observed with primary fibroblasts, these cytokines were not modulated in irradiated keratinocytes conditioned culture media (Supplementary Fig. S4). LPS-activated PBMCs cultured in conditioned media harvested from HKPM168, HKPM170 and HKPM172 cells produced IL-8 in addition to high levels of MCP-1, IL-6 and IL-1β in control (Supplementary Fig. S3) and irradiated keratinocyte supernatants. Surprisingly, given the absence of effects of HKPM168 supernatants on PBMCs inflammatory status, the LPS-induced production of IL-6, IL-8 and IL-1β was decreased in conditioned media from keratinocytes exposed to 2 Gy when compared to supernatants from control and/or low-dose irradiated cells, in supernatants collected at 48 h (IL-6, IL-1β) or at 24 and 48 h (IL-8) (Fig. 6). In contrast, conditioned culture media harvested from low-dose exposed keratinocytes could either enhance or reduce the production of cytokines by PBMCs following LPS stimulation. Supernatants collected 24 h after 50 mGy exposure enhance the LPSinduced IL-8 secretion by PBMCs, while those collected 48 h after 100 mGy exposure reduce LPS-induced IL-1β production. Thus, whereas exposure of keratinocytes to high-dose radiation impairs the response of PBMCs to LPS, the effects of low-dose radiation are mixed, with both potentiation and inhibition of cytokine secretion, when compared to non-irradiated keratinocytes. The overall result is a qualitatively different profile of cytokine secretion by PBMCs upon LPS stimulation.

DISCUSSION

We compared the cellular and bystander responses induced in/from primary fibroblasts and keratinocytes by low and high radiation doses with the aim to determine whether and how radiation exposure can modulate the functionality of immune cells. Our results show that radiation exposure elicits different patterns of responses in fibroblasts and keratinocytes, and that radiation-induced bystander signals have functional consequences as they affect the inflammatory status and response of purified PBMCs.

Radiation-induced modulation of gene expression in skin cells after low-and/or high-dose exposure have already been largely documented in primary fibroblasts [START_REF] Brackmann | Comparison of time and dose dependent gene expression and affected pathways in primary human fibroblasts after exposure to ionizing radiation[END_REF][START_REF] Ding | Gene expression profiles of normal human fibroblasts after exposure to ionizing radiation: a comparative study of low and high doses[END_REF][START_REF] Fujimori | Extremely low dose ionizing radiation up-regulates CXC chemokines in normal human fibroblasts[END_REF][START_REF] Warters | Differential gene expression in primary human skin keratinocytes and fibroblasts in response to ionizing radiation[END_REF], keratinocytes [START_REF] Warters | Differential gene expression in primary human skin keratinocytes and fibroblasts in response to ionizing radiation[END_REF][START_REF] Franco | Low-dose exposure to gamma rays induces specific gene regulations in normal human keratinocytes[END_REF] and skin/skin models [START_REF] Berglund | Transient genome-wide transcriptional response to low-dose ionizing radiation in vivo in humans[END_REF][START_REF] Goldberg | Human in vivo doseresponse to controlled, low-dose low linear energy transfer ionizing radiation exposure[END_REF][START_REF] Mezentsev | Global gene expression responses to low-or high-dose radiation in a human three-dimensional tissue model[END_REF][START_REF] Yunis | Genomic characterization of a three-dimensional skin model following exposure to ionizing radiation[END_REF]. Cell-cell signaling pathways are for example modulated in primary fibroblasts or skin plugs after low doses, while the response to higher doses was dominated by changes in cell proliferation and apoptosis pathways [START_REF] Ding | Gene expression profiles of normal human fibroblasts after exposure to ionizing radiation: a comparative study of low and high doses[END_REF][START_REF] Mezentsev | Global gene expression responses to low-or high-dose radiation in a human three-dimensional tissue model[END_REF][START_REF] Yunis | Genomic characterization of a three-dimensional skin model following exposure to ionizing radiation[END_REF]. In a different study, pathways related to cancer and metabolism were found induced only in high, but not low-dose exposed primary fibroblasts [START_REF] Brackmann | Comparison of time and dose dependent gene expression and affected pathways in primary human fibroblasts after exposure to ionizing radiation[END_REF]. Specific genes were found up-or down-regulated differently over time in low-dose exposed keratinocytes [START_REF] Franco | Low-dose exposure to gamma rays induces specific gene regulations in normal human keratinocytes[END_REF]. Collectively, these results illustrate the diversity and complexity of the transcriptional responses elicited by ionizing radiation in different experimental settings. In addition, these responses may also depend on inter-individual variability, as shown by the comparison of the response elicited in skin biopsies from different patients after in vivo exposure to 100 mGy, with as many as four out of eight patients showing no significant changes in the expression of genes belonging to the cytokine and inflammation pathways [START_REF] Goldberg | Human in vivo doseresponse to controlled, low-dose low linear energy transfer ionizing radiation exposure[END_REF]. Here, we compared the expression of a selected set of stress response and inflammatory genes in primary fibroblasts and keratinocytes established from the same donors after low-and high-dose exposure. While the relative expression of stress response genes was always up-regulated after high-dose exposure, expression of some of the inflammatory genes was unchanged, induced or repressed in the same samples (Figs 2 and4). It is unlikely that the reduced levels of IL-8 and TNFA transcripts in high-dose exposed fibroblasts and/or keratinocytes result from radiation-induced cell death, as, first, mortality never exceeds 20% after 10 Gy exposure, and second, in that case, a similar reduction would be observed for all genes analyzed. This different behavior in response to high-dose radiation exposure therefore suggests that the mechanisms responsible for the modulation of the two sets of stress and inflammatory genes are different in irradiated cells and are not governed solely by the activation of the ATM-dependent DDR.

The situation is however different in response to nutlin-3 stimulation, which results in the induction of these genes (Supplementary Fig. S1). These results strongly suggest that a strong and sustained activation of p53 activity is able to stimulate the transcription of inflammatory genes in primary fibroblasts and keratinocytes including CXCL8/IL-8, suggesting that its down-regulation in irradiated cells is not directly under the control of p53 activation. The level of p53 activation after exposure to 2 and 10 Gy might not be 'high' enough to promote a strong induction of inflammatory genes. They also suggest that beside the radiation-induced DDR, one or more additional mechanism(s) is/are induced that can repress inflammatory gene transcription or counteract p53 transcriptional activity in irradiated primary fibroblasts and keratinocytes. These mechanisms may for Fig. 5. Influence of BEs on PBMCs inflammatory status. The level of expression of IL1B, CCL3, CXCL8/IL8, CCNG1, DDB2 and PHPT1 genes was measured by RT-qPCR in purified PBMCs cultured 24 h in conditioned culture media harvested from non-irradiated and irradiated HFPM168 (top) and HKPM168 (bottom) cells harvested 24 h and 48 h post exposure. These volcano plots show the relative normalized expression of these 6 genes in PBMCs cultured in supernatants collected from cells exposed to 0.05 (left), 0.1 (center) and 2 (right) Gy, compared to their expression in non-irradiated control supernatants. The green and red lines represent a fold change (FC) of 1.6 time, respectively, and the blue line the threshold P-value = 0.05, calculated by a t-test. n = 3 for PBMCs cultured in each of eh HFMP and HKPM supernatants.

example involve the restraining of NF-κB activity by ATM [START_REF] Sabatel | Phosphorylation of p65(RelA) on Ser(547) by ATM represses NF-kappaBdependent transcription of specific genes after genotoxic stress[END_REF], which would occur concurrently of p53 activation by radiation. Irrespective of the exact nature of these mechanisms, it is interesting to note that the nutlin-3-induced transcriptional regulation of TGFB1, CXCL8/IL-8 and IL1B genes is different in HFPM and HKPM cells, illustrating here again differences in the regulation of their expression in primary fibroblasts and keratinocytes from a same donor. It will be of interest to identify other genes coding for immune factors similarly regulated, and the molecular mechanisms involved, to better understand the effects exposure and/or p53 activation on immune functions.

Different cytokines have been involved in the transmission of BEs [START_REF] Prise | Radiation-induced bystander signalling in cancer therapy[END_REF][START_REF] Lorimore | Chromosomal instability in unirradiated hemaopoietic cells induced by macrophages exposed in vivo to ionizing radiation[END_REF][START_REF] Shao | Role of TGF-β1 and nitric oxide in the bystander response of irradiated glioma cells[END_REF][START_REF] Wang | Radiation-induced bystander effect: early process and rapid assessment[END_REF].Our analysis of a limited number of selected cytokines illustrates qualitative and quantitative differences in cytokine production by primary fibroblasts and keratinocytes: MCP-1 and GM-CSF are selectively produced only by HFPM and HKPM cells, respectively; keratinocytes secrete more IL-8 than fibroblasts, and, conversely, fibroblasts produce more IL-6 than keratinocytes (Fig. 3). These differences may explain that the conditioned culture media harvested from fibroblasts and keratinocytes have different effects on the inflammatory status of bystander PBMCs, as only conditioned culture media harvested from irradiated fibroblasts down-regulate their expression of inflammatory genes. However, irradiated fibroblasts and irradiated keratinocytes supernatants are both able to regulate the secretion of inflammatory cytokines from LPS-activated PBMCs, with slightly different outcomes for IL-1β and IL-6. Furthermore, only keratinocytes, but not fibroblasts, support the secretion of IL-8 by LPSstimulated PBMCs. Thus, the secretory profiles of primary fibroblasts and keratinocytes are different, and BEs mediated by irradiated HFPM and HKPM cells on resting and activated immune cells are different and distinctly modulated by radiation exposure.

Supernatants from high-dose exposed HFPM and HKPM cells always reduce LPS-activated secretion of pro-inflammatory cytokines by PBMCs. In contrast, HKPM supernatants collected 24 h after exposure to 0.05 Gy induce a small but significant increase in LPS-induced IL-8 secretion (Fig. 6). High-dose-induced bystander signals appear to impair some aspects of the stimulation of PBMCs via activation of TLR4, the LPS receptor, while the effects of low-dose radiation can be more diverse.

Thus, bystander signaling may result in an imbalanced TLR4dependent inflammatory response, with an increase or a decrease of only selected cytokines. The inflammatory response is a highly organized, self-limiting process ending in a resolution phase which culminates in the restoration of tissue homeostasis. The transitions between inflammation initiation, amplification and resolution are orchestrated by successive waves of pro-inflammatory and anti-inflammatory, pro-resolving factors [START_REF] Medzhitov | Transcriptional control of the inflammatory response[END_REF][START_REF] Serhan | Resolution of in flammation: state of the art, definitions and terms[END_REF][START_REF] Serhan | Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators[END_REF]. In this context, bystander-induced qualitative and quantitative changes in the secretion of some, but not all, cytokines results in qualitative changes in the spectrum of secreted inflammatory mediators produced by activated immune cells. These changes may, in the long run, hamper the unfolding of the reaction resulting from TLR4 activation and modify or postpone its resolution, potentially resulting in un-resolved, inflammation or maladapted tissue homeostasis [START_REF] Feehan | Is resolution the end of inflammation?[END_REF]. These outcomes have been linked with numerous adverse conditions [START_REF] Balkwill | Smoldering and polarized inflammation in the initiation and promotion of malignant disease[END_REF], including the propagation of DNA damage to un-irradiated tissues following local irradiation in mice [START_REF] Lobachevsky | A functional immune system is required for the systemic genotoxic effects of localized irradiation[END_REF][START_REF] Redon | Tumors induce complex DNA damage in distant proliferative tissues in vivo[END_REF]. Radiation-induced BEs may therefore participate in the deleterious, unwanted effects of radiation exposure, including tumor-promoting inflammation [START_REF] Heeran | The radiation-induced bystander effect (RIBE) and its connections with the hallmarks of cancer[END_REF].

Previous work suggested that exposition of purified blood monocytes or mononuclear cells to low-and high-dose radiation could activate or potentiate TLR4-induced cytokine secretion [START_REF] El-Saghire | Low doses of ionizing radiation induce immune-stimulatory responses in isolated human primary monocytes[END_REF][START_REF] Menendez | The toll-like receptor gene family is integrated into human DNA damage and p53 networks[END_REF]. Our results now suggest that bystander signals received from both low-and high-dose exposed neighboring cells could modify the outcome of such TLR4 activation, for example after the migration of monocytes into tissues upon radiation injury. These interferences might for example impair the functional differentiation of monocytes into inflammatory macrophages and contribute to the creation of an inflammatory environment prone to support unwanted pathophysiological events [START_REF] Stoecklein | Radiation exposure induces inflammasome pathway activation in immune cells[END_REF]. However, at that time, we can only speculate on the eventual different outcomes of low-and high-dose elicited bystander signals.

It is generally admitted that low-dose radiation induce an inflammatory/tissue repair response whereas higher doses activate a p53dependent response [see for example, [START_REF] Mezentsev | Global gene expression responses to low-or high-dose radiation in a human three-dimensional tissue model[END_REF][START_REF] Menendez | The toll-like receptor gene family is integrated into human DNA damage and p53 networks[END_REF], and that bystander responses are most important after low to intermediate but not highdose exposure [START_REF] Lomax | Biological consequences of radiation-induced DNA damage: relevance to radiotherapy[END_REF][START_REF] Prise | Radiation-induced bystander signalling in cancer therapy[END_REF][START_REF] Portess | Low-dose irradiation of nontransformed cells stimulates the selective removal of precancerous cells via intercellular induction of apoptosis[END_REF][START_REF] Belyakov | Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts[END_REF]. This dichotomy probably raises from the fact that the cellular response to high-dose exposure is dominated by the activation of the ATM/p53 pathways during the DDR, which strongly determines cell fate and may therefore hinder the effects of bystander signals. In contrast, low-dose radiation may activate several types of stress responses, including inflammatory, oxidative, DNA damage and recovery/tissue repair responses that may be coordinated by different radiation-responsive transcription factors [START_REF] Mezentsev | Global gene expression responses to low-or high-dose radiation in a human three-dimensional tissue model[END_REF]. Cell fate is therefore the resultant of the interactions between these different pathways and, if none of them is overtly dominant [START_REF] Berglund | Transient genome-wide transcriptional response to low-dose ionizing radiation in vivo in humans[END_REF][START_REF] Mezentsev | Global gene expression responses to low-or high-dose radiation in a human three-dimensional tissue model[END_REF], the influence of modifiers released post radiation exposure, including cytokines, soluble mediators and microvesicles, is more easily evidenced. Here, supernatant transfer experiments allowed us to show that both low-and high-dose induced bystander signals elicited from irradiated primary fibroblasts and keratinocytes have functional consequences on PBMCs inflammatory status and activation. Furthermore, radiation-induced BEs do not result from a 'generic' radiation-induced cellular stress response, as they appear to be distinct for each cell lineage. Some of the mediators responsible for these effects are selectively produced, and, for those that are common, they are produced in different quantities and/or modulated with different kinetics and dose-response patterns in both cell types.

Fig. 1 .

 1 Fig. 1. Schematic representation of the experimental strategy designed to analyze the direct and bystander consequences of primary fibroblasts and keratinocytes exposure to low-and high-doses of γ -rays. Directs effects were analyzed by RT-qPCR and ELISA 24 h and 48 h after primary cells exposure (left). BEs were analyzed by RT-qPCR and ELISA after culture of resting or LPS-activated PBMCs in conditioned culture media (CM) harvested from control and irradiated primary cells.

Fig. 2 .

 2 Fig. 2. Modulation of stress and inflammatory gene expression in irradiated primary fibroblasts. The expression of stress (left) and inflammatory (right) genes was analyzed by RT-qPCR 24 h (top) and 48 h (bottom) after exposure of HFPM168 (n = 4), HFPM170 (n = 3) and HFPM172 (n = 4) cells to 0.05, 0.1, 2 and 10 Gy of γ -radiation. The x axis (Gy) indicates the dose of radiation. Only HFPM170 and HFPM172 were exposed to 10 Gy. For each gene, the level of expression in control and exposed cells was compared by an ANOVA test if the data were normally distributed or by a Kruskal-Wallis test, if they were not. When a value of P < 0.05 indicated significant differences in irradiated cells, pairwise comparisons were performed with a Tukey's test, after an ANOVA test, or a Dunn's post hoc test, after a Kruskal-Wallis test. The graphs represent mean ± SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001.

Fig. 6 .

 6 Fig. 6. Influence of BEs on PBMCs activation. Quantification of cytokine secretion by PBMCs stimulated by LPS (1 ng/mL) in control and irradiated primary fibroblasts (left) and keratinocytes (right). For HFPM cells, histograms represent the result of supernatant transfer experiments performed with conditioned culture media harvested 24 h and 48 h after exposure of HFPM168 (n = 3) and HFPM170 (n = 3 for IL-6 and n = 4 for IL-8 and MCP-1) to the indicated radiation doses. For HKPM cells, these experiments were performed with conditioned culture media harvested from HKPM168 (n = 3), HKPM170 (n = 3) and HKPM172 (n = 4) exposed to the same radiation doses. The x axis [Sn(Gy)] indicates the dose of radiation received by HFPM and HKPM cells before harvesting the conditioned culture medium. For each cytokine, the level of secretion in the different supernatants was normalized to that of PBMCs cultured in conditioned media harvested from corresponding non-irradiated HFPM or HKPM cells. Comparisons were performed by a Kruskal-Wallis test followed by pairwise comparisons by a Dunn's post hoc test when P < 0.05. The graphs represent mean ± SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001.

Downloaded from https://academic.oup.com/jrr/advance-article/doi/10.1093/jrr/rrac094/6993145 by CEA -Grenoble user on 06 March 2023

ACKNOWLEDGMENTS

This article is dedicated to the memory of our friend and colleague Catherine Aude-Garcia.

FUNDING

This work was supported by financing from Electricité de France (EDF) through the Comité de Programme Protection de l'Homme et de l'Environnement (CPPHE) -GGP V3-101 'Sciences du Vivant'; GRAL, a program from the Chemistry Biology Health (CBH) Graduate School of University Grenoble Alpes (grant number ANR-17-EURE-0003); and in part by the LABEX PRIMES (grant number ANR-11-LABX-0063).

SUPPLEMENTARY DATA

Supplementary data is available at RADRES Journal online.

CONFLICT OF INTEREST

The authors declare no conflict of interest.


María De Los

Angeles Bohórqu-Quintero

Daicy Yaneth Galv-Tarazona

Diana Marcela Arias-Moreno

Zaida Zarely Ojeda-Peréz

Sergio Ochatt

& Luis

Ernesto Rodríguez-Molano

Morphological and anatomical characterization of yellow diploid potato flower for effective breeding program

The diploid yellow potato (Solanum tuberosum L. Phureja Group) is an important plant genetic resource. In this study, we report for the first time the characterization of anther development and pollen formation in the cultivar Criolla Colombia. The description of morphological and histological characters of buds and flowers at different developmental stages permitted to identify ten main stages, from the differentiation of the male cells of the sporangium, meiosis, microspores formation and maturation, to the release of mature pollen. In addition, the results provide a graphic guide of the development of the anther, through the sequential and orderly formation of the epidermis, the endothecium, the middle layer and the nutritive layer or tapetum. This microanatomical information will be useful for work focused on androgenesis and identification of gene regulation in floral biology and gamete formation. Therefore, this study determined that to efficiently obtain haploids, flower buds between 5 and 8.9 mm long (stage 6 to 8) should be used, in which tetrads and microspores are in the early uninucleate and binucleate stage.

The Solanaceae family has been the center of important research advances for decades. It comprises 102 genera and 2500 species, with great abundance, distribution and endemism in Latin America [START_REF] Ortiz-Ramírez | Evolution of genes associated with gynoecium patterning and fruit development in Solanaceae[END_REF][START_REF] Mushtaq | Allelopathy in Solanaceae plants allelopathic Solanaceae plants[END_REF] . In the Solanaceae, the genus Solanum stands out, with approximately 2000 species registered including the potato, the main noncereal product commercialized globally [START_REF] Kazemiani | Shoot proliferation from potato (Solanum tuberosum cv. Agria) under different concentration of MS include vitamins and BAP medium[END_REF][START_REF] Álvarez | Identification of novel associations of candidate genes with resistance to late blight in Solanum tuberosum group Phureja[END_REF] . In Solanum, the Phureja group consists mainly of diploid potatoes. These show a wide phenotypic diversity and important agronomic and nutritional characteristics [START_REF] Álvarez | Identification of novel associations of candidate genes with resistance to late blight in Solanum tuberosum group Phureja[END_REF][START_REF] Piñeros-Niño | Hydroxycinnamic acids in cooked potato tubers from Solanum tuberosum group Phureja[END_REF] . Therefore, it is important to take advantage of its wild and commercial varieties in genetic improvement programs. The Creole potato or diploid yellow potato (Solanum tuberosum L. Phureja Group) [START_REF] Huamán | Reclassification of landrace populations of cultivated potatoes (Solanum sect. Petota)[END_REF] corresponds to morphotypes that present tubers with yellow skin color and flesh [START_REF] Rodríguez | Criolla Latina, Criolla Paisa y Criolla Colombia, nuevos cultivares de papa criolla para el Departamento de Antioquia (Colombia)[END_REF] , and it constitutes one of the most important plant genetic resources in Colombia, the largest producer, consumer and exporter of diploid potato in the world [START_REF] Herrera | Tecnologías de la Producción y Transformación de Papa Criolla[END_REF] , due to its nutritional and organoleptic value [START_REF] Peña | Nutritional contents of advanced breeding clones of Solanum tuberosum group Phureja[END_REF][START_REF] Mosquera | Breeding differently : Participatory selection and scaling up innovations in Colombia[END_REF] .

In Solanaceae, anther development and pollen grain formation have been described for Cestrum bigibbosum Francey [START_REF] Castro | Desarrollo de la antera y formación del grano de polen en Cestrum bigibbosum Francey (Solanace)[END_REF] , species of the genus Capsicum L. [START_REF] Magalhães | Meiotic irregularities in Capsicum L. species[END_REF] , Petunia hybrida Juss. [START_REF] Rezanejad | The response of anther and pollen development, pollen cellular material release and pollen proteins to air pollution in Petunia juss Juss. (Solanaceae). Iran[END_REF] , Solanum pimpinellifolium [START_REF] Qu | Cytological observation of Solanum pimpinellifolium L. Microspore development[END_REF] and potato, among others. In S. tuberosum, microsporogenesis is known in three tetraploid somatic hybrids and their di(ha) ploid crosses [START_REF] Pijnacker | Microsporogenesis in three tetraploid somatic hybrids of potato and their di(ha) ploid fusion partners[END_REF] . The meiotic behavior of pollen mother cells has also been reported for potato, in relation to the ploidy level of somatic hybrids (S. tuberosum × S. chacoense) [START_REF] Magalhães | Meiotic irregularities in Capsicum L. species[END_REF] , as also to the development of the anther, microsporogenesis and gametophyte formation [START_REF] Talebi | The study of anther development, microsporogenesis and formation of male gametophyte in potato (Solanum tuberosum L.)[END_REF] . However, these works are shallow and do not offer a detailed and extended descriptive and iconographic characterization of the process. As far as we know, no similar studies are known for creole potato.

It is essential to study the different aspects of pollen biology to understand the mechanisms of plant reproduction for a successful implementation of breeding programs, as they allow to increase the efficiency in the crossings, assuring an abundant production of seeds [START_REF] Dafni | Pollen viability and longevity: Practical, ecological and evolutionary implications[END_REF] . Pollen formation is a function of the stamen that takes place within the anther and involves numerous histochemical changes [START_REF] Fadón | Anther and pollen development in sweet cherry (Prunus avium L.) in relation to winter dormancy[END_REF]19 proceed in a sequential and orderly manner, and exhibit genetic and enzymatic coordination [START_REF] Agadi | Ontogeny of anther in Solanum viarum, Dunal[END_REF][START_REF] Aloisi | Polyamines in pollen: From microsporogenesis to fertilization[END_REF][START_REF] Zhang | Proteomes and phosphoproteomes of anther and pollen: Availability and progress[END_REF] . The analysis of the relationship between flower size and the stage of pollen development (microsporogenesis) provides an important tool for the induction of androgenesis [START_REF] Ochatt | Abiotic stress enhances androgenesis from isolated microspores of some legume species (Fabaceae)[END_REF] . This makes it possible to identify the stage or state in which the regeneration of haploid plants can be induced with greater efficiency [START_REF] Sharma | Orchid micropropagation: Regeneration competence of anther culture[END_REF][START_REF] Mishra | In-vitro androgenesis in rice: Advantages, constraints and future prospects[END_REF] . Doubled haploid production is a valuable biotechnology that can accelerate the breeding of new plant varieties by several years through the one-step creation of 100% homozygous plants [START_REF] Eliby | Developments and prospects for doubled haploid wheat[END_REF] . Haploids can be induced in vitro via cultivating the haploid gametes or in vivo through inter-and intraspecific hybridization [START_REF] Karimi-Ashtiyani | Centromere engineering as an emerging tool for haploid plant production: advances and challenges[END_REF] . In addition, it facilitates the estimation of the quantity and quality of the pollen, to optimize hybridization processes in genetic breeding programs [START_REF] Youmbi | Oil palm (Elaeis guineensis Jacq.) improvement: Pollen assessment for better conservation and germination[END_REF] . Therefore, the histological study of microsporogenesis offers opportunities for the control of fertility in plants of commercial interest [START_REF] Fernández | Anther and pollen development: A conserved developmental pathway[END_REF] . Additionally, knowing the tissue conformation of the anther and the general morphology of the pollen can have taxonomic and systematic utility [START_REF] Castro | Desarrollo de la antera y formación del grano de polen en Cestrum bigibbosum Francey (Solanace)[END_REF][START_REF] Talebi | The study of anther development, microsporogenesis and formation of male gametophyte in potato (Solanum tuberosum L.)[END_REF] .

There are studies on microsporogenesis [START_REF] Talebi | The study of anther development, microsporogenesis and formation of male gametophyte in potato (Solanum tuberosum L.)[END_REF] and pollen meiosis and mitosis in S. tuberosum [START_REF] Höglund | Meiosis in Solanum phureja[END_REF] . However, the different species and cultivars may present developmental particularities, which justifies the need for further research input on this subject [START_REF] Parra-Vega | Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.)[END_REF] . In diploid yellow potato the ontogeny and histological development of pollen and anther and their correlation with flower formation [START_REF] Agadi | Ontogeny of anther in Solanum viarum, Dunal[END_REF] still remain unknown, despite their commercial and agronomic relevance. Therefore, and considering that, the microspore is at the center of a variety of topics in modern plant science and breeding [START_REF] Touraev | The microspore: A haploid multipurpose cell[END_REF] , the aim of the present study was to characterize for the first time the pollen and anther formation, and to evaluate its association with anatomical changes in flower development in diploid yellow potato (S. tuberosum L. Phureja Group), cultivar Criolla Colombia; as a precise, fast, easy and reliable guide to identify microspores or pollen at development particular stages.

Results

Anatomical characterization of flower. Criolla Colombia presents perfect, actinomorphic, pedicelate, whorled and pentamerous flowers. The androecium contains five exserted stamens inserted in the corolla; which, has a rotated shape, predominant coloration of intense lilac and secondary white, on both sides. The ovary is superior, the stigma is greenish in color and protrudes from the anther ring (Fig. 1a). The flowers are distributed in a terminal cymose inflorescence. The anther contains four lobes that are attached to the filament by connective tissue and together form the stamen (Fig. 1b,c). We found that flower development can be associated with ten main stages (Fig. 1d,e), for buds between 0.1 and 15 mm.

Anther and pollen development.

Pollen development within the anther depends on joint interactions between gametophytic and sporophytic tissues. This process includes a series of crucial phases such as differentiation of male cells from the sporangium, meiosis, formation and maturation of the microspore, and finally release of mature pollen (Figs. 2, 3, and4).

We describe for the first time the correlation between bud/anther size and the progression of microspore development for cultivar Criolla Colombia. The landmark morphometric and histological macro-and microanatomical events for each developmental stage were determined (Table 1; Figs. 2, 3 and4). The data in Table 1 underline the relevance of undertaking the microscopic observations added to the phenological ones to ascertain the precise microsporogenetic stage of flower buds, given the small differences observed in their size. Additionally, the statistical analysis determined that these differences between the development stages for length and diameter of buds and anthers are significant (Kruskal-Wallis p < 2.2e-16; Table S1-S6).

Stage 0. Buds shorter than 1 mm and light green in color (Fig. 1d,e). At the histological level, primordia of sepals, petals, stamens, filaments, and ovary were observed and, in turn, dividing cells within these (Fig. 2-Stage 0). Within the primordium of the stamen, the preliminary formation of two cell layers and procambial cells was distinguished. L1, cells that will form the epidermis; L2, which will give rise to germ cells, endothecium, midline and tapetum, and procambial cells derived from a previously formed L3 layer, which will develop vascular and connective tissue [START_REF] Verma | Transcriptional regulation of anther development in Arabidopsis[END_REF][START_REF] Furuta | Molecular control of cell specification and cell differentiation during procambial development[END_REF] .

Stage 1.

Flower buds between 1 and 1.5 mm long and partially light green in color. Sepals larger than the buds (Fig. 1d,e). The anthers began to acquire a tetrasporangial shape, due to the continuous cell division in each of the lobes (Fig. 2-Stage1).

Stage 2. Bud length ranged from 1.6 to 2 mm. The bud was green and formation of petals was observed, below which the developing stamens are located (Fig. 1d,e). Sporangial cells covered the lobular space, and the epidermis, endothecium, medial layer, and tapetum were distinguished in the anther (Fig. 2a-Stage 2). In addition, sporophytic cells, located in the U-shaped region, were also identified.

Stage 3.

The buds were between 2.1 and 2.9 mm long and increased their volume. In addition, anthers covered by the greenish petals that were still united were observed, and early development of the gynoecium was observed (Fig. 1d,e). In the general structure of the bud, histologically, sepals, petals (located outside the stamens), anthers and ovary were distinguished. The cells of the sporangium were distinguishable (Fig. 2-Stage 3).

Stage 4.

Buds with a length between 3 and 3.9 mm. In this stage, an increase in the diameter of the bud and in the size of the anther, ovary, style, and stigma were observed. The union of the petals in the apical part was observed to be densely covered by trichomes (Fig. 1d,e). Histologically, the Pollen Mother Cells (PMC) began to separate from each other [START_REF] Mishra | In-vitro androgenesis in rice: Advantages, constraints and future prospects[END_REF] (Fig. 2-Stage 4).

Stage 5.

The length of the bud was between 4 and 4.9 mm. The base of sepals was colored with anthocyanins.

Larger petals, anthers and style were observed. The gynoecium acquired a whitish hue on the outside (Fig. 1d,e). The PMC began their first meiotic division, forming two dyads (chromosomal load n). Likewise, at this stage the septum and also amorphous tapetum cells were observed (Fig. 3-Stage 5).

Stage 6.

The buds were between 5 and 5.9 mm long, with thinner sepals towards the terminal part. The external-terminal coloration of the petals was whitish, and the length of the anthers increased (Fig. 1d,e). The Stage 7. The length of the buds was between 6 and 7.4 mm. An increase in the size of the anthers was observed, which covered the entire space of the bud. In the anther, the connective of the theca and the apical part of the sepals were demarcated, it was observed in purple color (Fig. 1d,e). From the tetrads, haploid microspores were released. In these, the formation of a large vacuole was observed and, in the periphery, primexine was observed, which serves as a scaffold for pollen wall formation [START_REF] Truskina | A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation[END_REF] (Fig. 3-Stage 7).

Stage 8. The buds measured between 7.5 and 8.9 mm. The size of the petals (whitish) became slightly larger than that of the sepals (dark green). The style and stigma showed white and green coloration, respectively. In the anthers (green-yellowish) an increase in size was noticeable (Fig. 1d,e). Histologically, the beginning of the resorption of the septum and the beginning of the integration of the two locules within each theca was observed. In microspores, the nucleus was displaced by the vacuole to one end and wall thickening occurred (Fig. 4-Stage 8).

Stage 9.

The length of the buds ranged from 9 to 10.9 mm. The petals showed lilac and white coloration in the basal and apical parts, respectively. The pistil and ovary increased in size. The color of anthers changed from green to yellow (Fig. 1d,e). Histologically, in the bilobed anther, the external locules (near the petals) were larger than the internal ones (near the pistil). In each of the haploid microspores formed during meiosis, the nucleus divided by asymmetric mitosis and developed a bicellular pollen grain, distinguishing the vegetative and germinative pores [START_REF] Magalhães | Meiotic irregularities in Capsicum L. species[END_REF] (immature male gametophyte) (Fig. 4-Stage 9).

Stage 10. Buds between 11 and 15 mm long, petals larger than the sepals and predominantly violet in color (with anthocyanin pigments). This stage occurs prior to anthesis, so the petals begin to separate at the apical part of the flower. The yellow anthers formed a ring on which the pistil was distinguished (Fig. 1d,e). In addition, anther dehiscence due to stomium cell degeneration was observed. Finally, three germinal openings were identified in the pollen grains and consequently their release occurred (Fig. 4-Stage 10). In addition, it was observed that in the diploid yellow potato crop, flowering is staggered and can last up to 80-85 days after planting. There are usually two flowering cycles; the first occurs on main stems, while the second is characteristic of secondary stems. Flower buds < 5 mm are identified approximately 28 days after emergence (crop emergence occurs approximately 20 days after tuber planting and corresponds to the appearance of the first leaves on the soil surface). While buds between 12 to 14 mm (stage 10) are observed 42 days after emergence (Fig. 5). An open flower can last 2-4 days depend on environmental conditions.

Finally, based on these observations, it could be suggested that the anther and pollen development period of diploid yellow potato involves 10 to 14 days (Fig. 5). This process is illustrated by means of a scheme that includes the main histological and cytological events associated with the anther and pollen development process (Fig. 5 and6). Considering the relation between the stages of development and the duration expressed in days.

Discussion

The characterization of anther and pollen development, as well as the mechanisms of pollen release, are essential to understand the formation of embryos and seeds in most angiosperm species [START_REF] Fernández | Anther and pollen development: A conserved developmental pathway[END_REF][START_REF] Zhang | Specification of tapetum and microsporocyte cells within the anther[END_REF] . In this study, it was shown that the morphological and descriptive characterization of the flower for cultivar Criolla Colombia agree with those described for different Solanum species, suggesting a pattern of similar correspondence within the genus [START_REF]Recopilación de la Investigación del Sistema Productivo Papa criolla[END_REF] .

Pollen is the male gametophyte that carries plant genetic information and its study is relevant since the adequate male germline development is a prerequisite for proper crops performance [START_REF] Lora | Pollen development and viability in diploid and doubled diploid citrus species[END_REF][START_REF] Wang | Microsporogenesis, pollen ornamentation, viability of stored Taxodium distichum var. distichum pollen and its feasibility for cross breeding[END_REF] . Pollen development is a critical step in plant development that is needed for successful breeding and seed formation [START_REF] Fernández | Anther and pollen development: A conserved developmental pathway[END_REF] . In this study, the microsporogenesis from the differentiation of the archeosporial cells to the maturation of the pollen was described for the cultivar Criolla Colombia through ten stages. This process has also been referenced for other Solanaceae such as cherry tomato (Solanum pimpinellifolium) [START_REF] Qu | Cytological observation of Solanum pimpinellifolium L. Microspore development[END_REF] , tomato (Solanum lycopersicum) [START_REF] Niazian | Modeling callus induction and regeneration in an anther culture of tomato (Lycopersicon esculentum L.) using image processing and artificial neural network method[END_REF] and tobacco (Nicotiana tobaccum) [START_REF] Yu | Reactive oxygen species mediate tapetal programmed cell death in tobacco and tomato[END_REF] , in terms of the formation of anthers and male gametophytes.

Like other angiosperms, in the development of Criolla successful anther/microspore culture largely depends on the use of microspores at appropriate developmental stages at the time of culture, which can be specific for each plant species and genotype [START_REF] Adhikari | Association of floral bud and anther size with microspore developmental stage in campari tomato[END_REF] . Here, the correlation between morphological characteristics, such as floral bud length and diameter and anther length, and specific microspore developmental stages is described for the first time, for cultivar Criolla Colombia. The findings of the present work suggest that flower buds with lengths between 5 and 8.9 mm (stage 6), in which tetrads and microspores are in the uninucleate and early binucleate stages, should be used for the induction of androgenesis, as also observed in several other species [START_REF] Parra-Vega | Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.)[END_REF][START_REF] Niazian | Modeling callus induction and regeneration in an anther culture of tomato (Lycopersicon esculentum L.) using image processing and artificial neural network method[END_REF][START_REF] Seguí-Simarro | Androgenesis in Solanaceae[END_REF][START_REF] Vural | Development of androgenesis studies on eggplant (Solanum melongena L.) in Turkey from past to present[END_REF] . In this study, these cytological phases were observed within the characterization of the formation of male gametes, for the first time in Solanum tuberosum L. Phureja. So, towards the middle of the process, the first meiotic division occurred and with it the formation of dyads; followed by meiosis II, which gave rise to the development of tetrads (stage 6 to 8). Subsequently, the presence of vacuolated haploid microspores (stage 7) that underwent mitosis, giving rise to immature gametophytes (stage 8), was distinctive. In addition, in the last stages, the typical morphology of the pollen was identified, with its corresponding germinative pores.

In Criolla Colombia, pollen formation began with the differentiation of archeosporial cells into primary parietal and sporogenous cells [START_REF] Chaudhry | Reproductive Biology of Angiosperms[END_REF] ; which gave rise to the four typical lobes of the anther morphology. Within each lobe, the epidermis, the endothecium, the middle layer and the tapetum were identified. These layers play important roles in microsporogenesis. Thus, for example, the epidermis provides structural support, protects and prevents water loss in the anthers. The endothecium, together with the epidermis, increases the thickness of the outer wall of the lobe and provide mechanical force for the dehiscence of the anther [START_REF] Talebi | The study of anther development, microsporogenesis and formation of male gametophyte in potato (Solanum tuberosum L.)[END_REF] . The middle layer completely surrounds the pollen sac and, together with the tapetum, performs secretory and nutritional functions during pollen development [START_REF] Talebi | The study of anther development, microsporogenesis and formation of male gametophyte in potato (Solanum tuberosum L.)[END_REF] .

The histological micropreparations obtained here also allowed to identify the septum, a layer that separates the locules within each theca in the anther, and whose reabsorption gave rise to the integration of a single locule per theca. Likewise, in this study the intine and exine pollen layers were notable in haploid microspores and mature pollen, respectively. On the other hand, it was distinctive that with the reabsorption of the stomium, the anther acquired the anatomical characteristics necessary for its opening and the subsequent release of mature pollen [START_REF] Åstrand | Evolution and diversity of the angiosperm anther: Trends in function and development[END_REF] .

Additionally, it is important to note that the observations allowed to identify microspores in different stages of development within and between anthers. So that, the processes involved in microsporogenesis may not be synchronous within a flower, an anther, and even a microsporangium [START_REF] Adhikari | Association of floral bud and anther size with microspore developmental stage in campari tomato[END_REF] . Asynchrony appears to be normal and has been documented for several species [START_REF] Parra-Vega | Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.)[END_REF][START_REF] Adhikari | Association of floral bud and anther size with microspore developmental stage in campari tomato[END_REF][START_REF] Sato | Anatomy and development of the staminate flower, microsporogenesis and microgametogenesis of species of Lophophytum (Balanophoraceae) in[END_REF] . This could cause metabolic differences between developing microspores and thus physiologically heterogeneous pollen grains [START_REF] García | It is a matter of timing: Asynchrony during pollen development and its consequences on pollen performance in angiosperms-A review[END_REF] . These events may have an adaptive role, since more efficient pollen grains would be selected in terms of homeostasis during pollen tube growth, tolerance to desiccation, resilience, speed of (re)hydration and germination [START_REF] García | It is a matter of timing: Asynchrony during pollen development and its consequences on pollen performance in angiosperms-A review[END_REF] .

Likewise, the histological events of anther and microspore formation characterized in this study, agree with those described for different Solanum species [START_REF] Qu | Cytological observation of Solanum pimpinellifolium L. Microspore development[END_REF][START_REF] Pijnacker | Microsporogenesis in three tetraploid somatic hybrids of potato and their di(ha) ploid fusion partners[END_REF][START_REF] Talebi | The study of anther development, microsporogenesis and formation of male gametophyte in potato (Solanum tuberosum L.)[END_REF][START_REF] Salas | Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures[END_REF] . This research consolidated a precise and non-invasive method to correlate morphological markers with microspore/pollen grain formation stages for cultivar Criolla Colombia. This will guide the selection and collection of flower buds/anthers harboring microspores at particular stages of development [START_REF] Parra-Vega | Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.)[END_REF][START_REF] Adhikari | Association of floral bud and anther size with microspore developmental stage in campari tomato[END_REF][START_REF] Adhikari | Association of floral bud and anther size with microspore developmental Campari tomato[END_REF] . Therefore, this new knowledge should be used within in vitro culture for the isolation, storage and use of anthers or microspores, for the evaluation of pollen viability or vigor and/or for obtaining double haploids 19 . It is well known that, within breeding programs, obtaining double haploids is one of the greatest limitations [START_REF] Lindhout | Towards F1 hybrid seed potato breeding[END_REF] , and these findings would allow its accelerated improvement to efficiently obtain diploid, self-compatible and 100% homozygous plants [START_REF] Du | Developing a new model system for potato genetics by androgenesis[END_REF] .

This research describes jointly and for the first time the development of the buds, the anther, and the pollen in potatoes. As such, the information presented in this study is of great relevance not only for creole potato crop but also for other commercially important Solanaceae. The results obtained are expected to strengthen the fundamental and applied agronomic research as well as commercial growth of this important phytogenetic resource, of high internal demand and great export potential [START_REF] Sierra | Detección molecular de seis virus de Arn en brotes de tubérculos de papa criolla (Solanum phureja) en Antioquia, Colombia[END_REF] .

Conclusion

This work offers the first macro-and micro-anatomical report of anther and pollen development in Solanum tuberosum L. Phureja, in general, and for Criolla Colombia potato in particular. Our results extend the knowledge on the identity and differentiation of microsporogenesis in this species of great economic importance. The histo-cytological events of the ten stages of pollen and anther development detailed here could be the basis for the formulation of identification studies on gene regulation in floral biology and gamete formation, as well as to obtain haploids and double haploids of utility in breeding programs.

Methods

Plant material. Solanum tuberosum L. Phureja Group, Cultivar Criolla Colombia, is a native cultivar type, the result of a clonal selection process within the population of genotypes with yellow round tuber [START_REF] Rodríguez | Criolla Latina, Criolla Paisa y Criolla Colombia, nuevos cultivares de papa criolla para el departamento de Antioquia (Colombia)[END_REF] .

The research was carried out at the BIOPLASMA Plant Tissue Culture Laboratory of the Faculty of Science within the Universidad Pedagógica y Tecnológica de Colombia. All procedures were conducted in accordance to the guidelines. For this study, samples of plant material were supplied by Luis Ernesto Rodríguez-Molano researcher who obtained the registration of the cultivar criolla Colombia as member of the potato research group of Faculty of Agronomy, within the Universidad Nacional de Colombia.

Inflorescences with buds and flowers in different stages of development were stored immediately maintaining the cold chain (5 °C) from the separation of the plant to their processing in the laboratory. Buds, flowers, and anthers representative of each developmental stage were photographed for illustrative purposes. In addition, to know the time required by flower buds to convert from one stage to another, we follow the flowering process in the crop.

Anatomical and metric characterization of the flower. The floral development in diploid yellow

potato cultivar Criolla Colombia was described from the appearance of flower buds to full bloom (more than 75% of open flowers), using qualitative descriptors proposed by Navarro et al. (2010) [START_REF] Navarro | Caracterización morfoagronómica y molecular de 19 genotipos de papa guata y chaucha (Solanum tuberosum L. y Solanum phurejajuz et Buk) cultivados en el departamento de Nariño[END_REF] and Gómez (2000) [START_REF] Gómez | Guía para las caracterizaciones morfológicas básicas en colecciones de papas nativas[END_REF] as a guide. These included degree of flowering, shape of the corolla, color of the flower, predominant color, intensity of predominant color, secondary color, distribution of secondary color, pigmentation in anthers, pigmentation in the pistil, color of the calyx and color of the pedicel.

The length and diameter of buds (n = 250) and anthers (n = 250) were measured. The results for each stage of development were expressed as the mean ± standard deviation. Given that not all data showed a normal distribution, the non-parametric Kruskal-Wallis (P < 0.05) test followed by Dunn's test was used, instead of analysis of variance (ANOVA), to compare the stages for each variable measured. Histological characterization. For each developmental stage, anthers were isolated and fixed for 24 h in FAA (50% ethanol, 5.0% glacial acetic acid, 3.7% formaldehyde). Subsequently, the samples were dehydrated in a series of ethanol (50, 60, 70, 80, 96, 3 × 100 v/v, 3 h per step), isopropanol and acetone (100 v/v, 1 h). Flower buds and anthers were embedded in paraffin, and histological sections (8-12 µm) were obtained with a rotary microtome. Cross-sections were stained with Safranin-FastGreen and observed under a microscope. These were selected and used in the microstructure analysis. The stages of microspore/pollen development were characterized according to reference studies 14,16,57,58. . For each stage twenty micropreparations (technical replicates) were observed, the best being selected to graphically represent the development of microsporogenesis.

Statement stating procedures according on plant ethics. All procedures were conducted in compliance with specific norms and regulations. For this study, samples of plant material were supplied by Luis Ernesto Rodriguez-Molano researcher (co-author of this paper) who obtained the registration (PAP-05-39) of the cultivar criolla Colombia as member of the potato research group of Faculty of Agronomy, within the Universidad Nacional de Colombia. The registration and complementary information related to this cultivar can be consulted through the link https:// www. papau nc. com/ catal ogo/ criol la-colom bia or in book [START_REF] Eduardo | Papa criolla (Solanum tuberosum Grupo Phureja): Manual de Recomendaciones Técnicas para su Cultivo en el Departamento de Cundinamarca[END_REF] and article [START_REF] Rodríguez | Criolla Latina, Criolla Paisa y Criolla Colombia, nuevos cultivares de papa criolla para el departamento de Antioquia (Colombia)[END_REF] .

Figure 1 .

 1 Figure 1. Buds and flowers of diploid yellow potato (Solanum tuberosum Grupo Phureja) cultivar criolla Colombia (scale bar a-d = 5 mm; e = 1 mm). Arabic numerals 1 to 10 represent anther developmental stages. Representative images of: (a) Perfect flowers with sepals, petals, anther ring and stigma. (b) Anthers. (c) Androecium and Gynoecium. (d) Development of buds from state 0 to 10. (e) Full bud and its cross section developing from state 0 to 10. ia immature anther, ma mature anther. Photographs and images created by the authors.

Figure 3 .

 3 Figure 3. Anther development and pollen formation in diploid yellow potato (Solanum tuberosum Phureja Group) Criolla Colombia cultivar. States of development: 5-7. Dy Dyads, E epidermis, En endothecium, Msp microspore, Prx primexin, St septum, T tapetum, Tds tetrads, V vascular tissue. Scale bars, 100 µm (column 1); 50 µm (column 2); 25 µm (column 3). Image created by the authors.

Figure 4 .

 4 Figure 4. Anther development and pollen formation in diploid yellow potato (Solanum tuberosum Phureja Group) Criolla Colombia cultivar. States of development: 8-10. E epidermis, Ex exine, Msp microspore, PG pollen grain, Sr stomium region, St septum, V vascular tissue. Scale bars, 100 µm (column 1); 50 µm (column 2); 25 µm (column 3). Image created by the authors.

Figure 5 .

 5 Figure 5. Schematic view of formation of diploid yellow potato anther cell layers. The diagram shows the relationship between the stages of flower bud development and the duration expressed in days. The left side of the figure details the first six stages (0 to 6) of anther development; while the right side of the image shows stages 7 to 10. D Dyads, E epidermis, En endothecium, L1, L2 lines of primordia of the stamen, Ml midline, Msp microspore, Pc procambial cells, PG pollen grain, Pl parietal line, PMC pollen mother cell, Sc sporangial cells, Sr stomium region, St septum, T tapetum, Tds tetrads, V vascular tissue. Asterisk: corresponds to the number of days after emergence. Scheme devised and recreated by the authors with images illustrated by Camila Reyes and Angie Carvajal.

Figure 6 .

 6 Figure 6. Schematic view of cytological events associated with development of bud, anther y pollen in diploid yellow potato. Scheme devised and recreated by the authors with images illustrated by Camila Reyes and Angie Carvajal.

Table 1 .

 1 Stages of anther and pollen development in diploid potato cultivar Criolla Colombia (Solanum tuberosum Phureja Group). The results showed significant differences between all the development stages, for length and diameter of buds and anthers, according to Kruskal-Wallis and Dunn's tests. SD standard deviation.

	Stage

Bud Anther Landmark stage of development Length X ± SD (mm) Length X ± SD (mm) Diameter X ± SD (mm)

	0	0.912 ± 0.040	0.225 ± 0.038	0.086 ± 0.008	Primordia
	1	1.425 ± 0.125	0.488 ± 0.105	0.260 ± 0.022	Formation of anther lobes and primary parietal and sporangia cells
	2	1.842 ± 0.115	0.796 ± 0.158	0.454 ± 0.037	Layers: epidermis, endothecium, midline, and tapetum
	3	2.666 ± 0.175	1.566 ± 0.271	0.652 ± 0.024	Sporangial cells
	4	3.616 ± 0.215	2.411 ± 0.147	0.859 ± 0.023	Pollen mother cell
	5	4.596 ± 0.303	2.755 ± 0.090	1.039 ± 0.022	Dyads
	6	5.618 ± 0.205	3.566 ± 0.205	1.256 ± 0.026	Tetrads
	7	7.093 ± 0.234	4.445 ± 0.258	1.456 ± 0.024	Young microspores
	8	8.232 ± 0.366	5.458 ± 0.264	1.664 ± 0.022	Late microspores
	9	10.173 ± 0.442	6.330 ± 0.121	1.844 ± 0.022	Mature pollen
	10	13.308 ± 1.156	6.864 ± 0.095	2.052 ± 0.021	Anther dehiscence: pollen release

Scientific Reports | (2022) 12:16402 | https://doi.org/10.1038/s41598-022-20439-6

Acknowledgements

The authors thank the Ministry of Science, Technology, and Innovation of Colombia, the Government of Boyacá, Boyacá and Colombia Bio Program and the Universidad Pedagógica y Tecnológica de Colombia, for financing the project entitled "Implementation of biotechnological and agricultural processes for the cultivation of clean species of ancestral varieties of potato (Solanum sp.) from the agro-ecosystems of the department". Convocation 794 of 2017 R&D projects for the technological development of a biological origin in the Department of Boyacá. Additionally, the authors thank the BIOPLASMA-UPTC research group and exalt the contribution of Dr. José Constantino Pacheco Maldonado † who also conceived and designed the research.

Data availability

The datasets generated and/or analyzed during the current study are available online in "Link Supporting data-Bohórquez et al. 2022".

Competing interests

The authors declare no competing interests.