Passet, B., Rigourd, V Ludivine Doridot

Bruno Passet

Virginie Rigourd

Céline Méharts

Sandrine Barbaux

Aurélien Ducat

Françoise Mondon

Marthe Vilotte

Johann Castille

Michelle Breuiller-Fouché

Céline Méhats

Michelle Breuiller- 5 Fouche

Nathalie Daniel

Fabienne Le Provost

Anne-Laure Bauchet

Véronique Baudrie

Alexandre Hertig

Christophe Buffat

Umberto Simeoni

Guy Germain

Jean-Luc Vilotte

Daniel Vaiman
email: daniel.vaiman@inserm.fr

Preeclampsia-like symptoms induced in mice by feto-placental expression of STOX1 are reversed by aspirin treatment

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proteinuria during gestation and responsible for maternal and foetal morbi-mortality. STOX1, encoding 32 a transcription factor, was the first gene identified by positional cloning approaches as associated with 33 preeclampsia. Its overexpression in choriocarcinoma cells mimics the transcriptional consequences of 34 preeclampsia in the human placenta. Here we created transgenic mouse strains overexpressing human 35 STOX1. Wild-type female mice crossed with transgenic STOX1-males reproduce accurately the 36 symptoms of severe preeclampsia: gestational hypertension, proteinuria, and elevated plasma level of 37 sFlt1 and sEng. Placental and kidney histology were altered. All the symptoms are prevented or 38 alleviated by aspirin treatment. STOX1-overexpressing mice constitute a unique model for studying 39 preeclampsia, will allow testing therapeutic approaches, and assessing the long term effects of the 40 preeclamptic syndrome. 41 Keywords: hypertension, mouse model, preeclampsia, proteinuria, STOX1, 42

Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression 3 Preeclampsia (PE) is a pervasive complication of human pregnancy characterized by a 44 gestational hypertension associated with proteinuria occurring from mid-gestation [START_REF] Sibai | Thrombophilia and severe preeclampsia: time to screen and treat in future 296 pregnancies?[END_REF] . Worldwide, this 45 syndrome affects ~5% of pregnant women, and is a leading cause of maternal mortality, especially in 46 low and middle income countries [START_REF] Firoz | Pre-eclampsia in low and middle income 298 countries[END_REF][START_REF] Goldenberg | Lessons for low-income 300 regions following the reduction in hypertension-related maternal mortality in high-income 301 countries[END_REF] ; it is also a direct cause of iatrogenic prematurity [START_REF] Goldenberg | Epidemiology and causes of preterm birth[END_REF] , since the only 47 definitive therapeutic act is the extraction of the foeto-placental unit . 48 STOX1 is a transcription factor belonging to the enlarged Forkhead Box gene family, that has 49 been associated with preeclampsia [START_REF] Van Dijk | Maternal segregation of the Dutch preeclampsia locus at 10q22 with a new 306 member of the winged helix gene family[END_REF] , able to modulate trophoblast proliferation and migration [START_REF] Van Dijk | The 308 STOX1 genotype associated with pre-eclampsia leads to a reduction of trophoblast invasion 309 by alpha-T-catenin upregulation[END_REF] . 50 STOX1 is maternally expressed in a specific cell type of the placenta, column extravillous 51 trophoblasts [START_REF] Van Dijk | Differential methylation of STOX1 in human placenta[END_REF] . Overexpression of STOX1 in human choriocarcinoma cells induces transcription 52 alterations that mimic those of preeclamptic placentas [START_REF] Rigourd | STOX1 overexpression in choriocarcinoma cells mimics 314 transcriptional alterations observed in preeclamptic placentas[END_REF][START_REF] Fenstad | STOX2 but not 317 STOX1 is differentially expressed in decidua from preeclamptic women[END_REF] . Founds and coworkers showed in a 53 transcriptomic analysis that STOX1 is overexpressed (x 2.1, p = 0.013) during the first trimester in 54 pregnancies that will have a preeclamptic outcome [START_REF] Founds | Altered 320 global gene expression in first trimester placentas of women destined to develop preeclampsia[END_REF] . 55

In the present work, we generated mice overexpressing human STOX1. Wild-type female mice 56 crossed with transgenic males overexpressing STOX1, have a severe gestational hypertension, 57 proteinuria, an increased plasma level of soluble antiangiogenic factors as well as kidney and placenta 58 histological alterations. We also demonstrate a beneficial effect of low-dose aspirin treatment on the 59 maternal symptoms. Hence, STOX1 overexpressing mice constitute a unique model for studying 60 severe preeclampsia. 61 62 Methods 63 Some more details of the methods are provided as supplemental data. 64

Animals and Trangenesis and aspirin treatment 65

The complete ORF of the human STOX1 (A isoform) cDNA was used to generate transgenic 66 animals [START_REF] Vilotte | Efficient tissue-specific 323 expression of bovine alpha-lactalbumin in transgenic mice[END_REF] . The number of copies of the transgene was assessed by qPCR from the mouse DNA 67 relative to a single copy gene. Transmission analysis through generations showed on the three strains 68 that were analyzed that there was a single locus of insertion.

Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression 4 Animals (FVB/N) were bred in the animal facility of INRA (Jouy en Josas, France) in a 70 controlled environment (light/dark cycle, temperature, free access to food and water). Blood 71 collection, urine collection, blood pressure measures and kidney perfusion were performed according 72 to standardized protocols, as well as supplementation of aspirin in the drinking water (See 73 supplemental methods) 74

RNA extraction and Quantitative RT-PCR conditions 75

Total RNA from collected tissues was extracted and quantified by spectrophotometry. Quality 76 of the RNA was systematically verified by Bioanalyzer and RIN consistently > 8. After reverse 77 transcription, qRT-PCR was performed. The efficiency of the PCR was checked for every primer 78 couple and estimated (Supplemental Methods). The Cts were normalized by the Ct values obtained for 79 the murine succinate dehydrogenase subunit A (Sdha) used as a reporter gene and shown previously to 80 be stable and highly expressed in the placenta [START_REF] Meller | Evaluation of housekeeping genes in 325 placental comparative expression studies[END_REF] . 81 All primers are presented as Supplemental Table 1. 82

Blood pressure measurements 83

Blood pressure was measured repeatedly in the tail artery in mice before, during and after pregnancy 84 (for a total of around 35 consecutive days). Measurements were performed using a computerized, non-85 invasive tail-cuff plethysmography method (Letica 5001, Bioseb), using thermostatically warmed 86 restrainers designed for mice and adapted to the size of the animal. This system uses volume-pressure 87 recording technology to detect changes in tail volume that corresponds to systolic and diastolic 88 pressures during each measurement cycle. Unanesthetized mice previously accustomed during one 89 week to the manipulation (systolic blood pressure stabilized), were placed in plastic holders and our 90 protocol consisted of at least 5 acceptable measurements daily, which means without detectable 91 movement of the mice. Thirty-six gestations were followed, which represents more than 5000 92 independent measurements of blood pressure by two independent researchers (systolic, diastolic and 93 median always displayed a highly similar profile). In addition, invasive measures were performed on 94 14 WT mice mated with WT or transgenic males at the end of gestation (14.5 to 17,5 dpc). The Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression 5 catheter was inserted into the left common carotid artery under xylazine/ketamine anesthesia. Mice 96 were kept on a heating pad during the experimentation. Blood Pressure was recorded and analyzed 97 from continuous measurements using BIOPAC MP36 system. The extraction of the Systolic Blood 98 Pressure (SBP) started when cardiovascular parameters were stable. 99

Assessment of Albumin/creatinine ratio (ACR) 100

Urine samples were collected daily during gestation and were pooled at early (1-5 dpc), 101 middle (9-12 dpc) and late (15-18 dpc) gestation times separately for each mice. Albumin-to-102 creatinine ratio was measured in these urine specimens using the kit ELISA of Exocell (Philadelphia, 103 PA). 104 sFlt1 and sEng plasmatic concentration assessment 105 Enzyme-linked immunosorbent assays were used to determine the concentration of sFlt1 and 106 sEng in mouse plasma with commercial kits (R&D Systems). 107

Histological analysis 108

Placentas and kidneys were sampled from 10 transgenic and WT mice (after heart perfusion 109 by 20 ml of PBS in the case of the kidneys). Anti-fibrin immunostaining of slides from paraffin-110 embedded kidneys was performed using the anti-human fibrinogen at 1/400 from Dako (A0080) and 111 the LSAB revelation system (Dako, K0679) according to the manufacter's instructions (standard 112 conditions). Placental histology was carried out after HES or PAS staining. The number of vacuolated 113 foci present in the labyrinth was counted after HE staining, a 1-5 grade was given according to the 114 level of PAS intensity, and the PAS positive areas were measured in the labyrinth. 115

Statistical analysis 116

During this study, parametric approaches were used using ANOVA followed by post-hoc 117 The expression levels of human and mouse STOX1 were tested by qRT-PCR in the kidney, 129 liver, brain and placenta of pregnant TgSTOX13 and TgSTOX42 females, as well as in the testis and 130 spermatozoa of transgenic males of both strains, using species-specific primers (Table 1); hSTOX1 131 expression was comparatively higher in placental tissue than in any other tissue or in spermatozoa. 132 qRT-PCR efficiency was maximal for mouse and human STOX, allowing to suggest that the placental 133 expression level of the human hSTOX1 was roughly similar to that of mStox1 in TgSTOX13 134 transgenic mice (Table 1) and about 13 fold higher in TgSTOX42. In the other tissues tested (kidney, 135 brain, testis and liver) the transgene expression level was lower than that of the endogenous gene, but 136 always higher in TgSTOX42 than in TgSTOX13 (Table 1). Since there is a unique transgene insertion 137 in both strains (as tested by quantitative PCR), we assume that the difference in the expression level 138 was strictly regulated by the genomic position of this insertion In transgenic placentas from 139 WTxTgSTOX42 crosses, where the transgene expression is the highest, the endogenous gene was 140 overexpressed 4 fold (p = 2.10 -11 by Student test compared to WTxWT crosses). 141

STOX1 transgenic mice develop a strong gestational hypertension, proteinuria, and

142 present an increased level of circulating sFlt1 and sEng.

143 Wild type females were crossed with males of three genotypes (WT, TgSTOX13 or 144 TgSTOX42) to ensure that the transgene expression be restricted to the foeto-placental unit. Blood Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression 7 pressure was monitored daily in the various crosses (Figure 1A). Increase in blood pressure during 146 gestation was observed in all the cases of crosses involving transgenic males. The blood pressure 147 curves reached a plateau in both strains around E10.5, until days 14.5-15.5, when the blood pressure 148 tends to increase again of an additional 15-20 mm Hg. The maximal increase reached about 80 mm Hg 149 from a normal pressure of ~116 mm Hg (Figure 1A). Blood pressure returned to normal rapidly (in 150 about 48 hours) after parturition. In addition, we carried out measurements by catheterization of one 151 of the carotid at late gestation times, and consistently with human blood pressure data under anesthesia 152 13 , the increase in mice carrying transgenic fetuses was present, albeit reduced; the blood pressure was 153 12 mmHg more elevated in the 'transgenic' crosses (p=0.008, Supplementary Figure 1). 154

Proteinuria was evaluated as a ratio between albumin and creatinin (ACR) (Figure 1B). After 155 individual normalization, we observed an increase of the ACR comprised between ~1.7 to 1.9 at mid 156 gestation (p=0.02 and 0.03 for TgSTOX13 and tgSTOX42, respectively) and between ~1.8 to 3.8 fold 157 at late gestation points (p=0.026 and 0.002, respectively), in females crossed with transgenic males 158 compared to control crosses. 159 sFlt1, considered as a biomarker for preeclampsia in the human disease [START_REF] Maynard | Excess placental soluble fms-331 like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and 332 proteinuria in preeclampsia[END_REF][START_REF] Levine | Circulating angiogenic 335 factors and the risk of preeclampsia[END_REF][START_REF] Hu | 337 Subfertility and defective folliculogenesis in female mice lacking androgen receptor[END_REF] was monitored 160 (Figure 2A). The average concentration in the plasma of pregnant mice was 2075 pg/ml when 161 TgSTOX13 males have fathered and 2455 pg/ml when crossed with TgSTOX42 males, an increase of 162 1.9 and 2.3 fold compared to control (1076 pg/ml), respectively (p = 0.02 and 0.03). Soluble endoglin 163 (sEng) was also measured in the plasma (Figure 2B), and found increased in the crosses involving 164 34.0 ± 11.8 in WT, Figure 3A, p = 3,4 10 -3 and p = 6.4 10 -7 respectively), because of a mild mesangial 172 cell proliferation. Immunohistochemistry revealed subendothelial fibrin deposition, with a 173 predominantly peripheral pattern. These changes are strongly reminiscent of the renal pathology 174 observed in human preeclampsia [START_REF] Gaber | Renal pathology in pre-eclampsia[END_REF][START_REF] Fogo | Renal diseases in pregnancy[END_REF][START_REF] Fiaschi | The histopathology of the kidney in toxaemia. Serial renal biopsies 343 during pregnancy, puerperium, and several years post-partum[END_REF] and could not be observed in the kidneys of WT pregnant mice 175 crossed with WT males (Figure 3B upper and middle photographs). No major abnormality was found 176 in the tubulo-interstitial compartment, or in the larger vessels, in either group. 177

Aspirin treatment efficiently improves the maternal phenotype.

178 WT females crossed with TgSTOX13 or TgSTOX42 males were treated with aspirin at low 179 doses in the drinking water during the whole gestation. As a control, WT pregnant females received 180 aspirin all along their pregnancy after a cross with WT males. Aspirin completely prevented the onset 181 of hypertension and brought the ACR to normal values at mid and late gestation times (Figures 1A and182 1B). Fibrin immunolabeling of the kidneys showed that the deposit was considerably reduced in 183 aspirin-treated wild type females crossed with TgSTOX13 or TgSTOX42 males, compared with non 184 treated females (Figure 3B). 185

STOX1 transgenic placentas present structural alterations.

186

The anatomical alterations of hemizygous TgSTOX13 and TgSTOX42 (validated as 187 transgenic by PCR) placentas were studied by histology (Figure 4A). No difference was observed in 188 gross morphology at 16.5 dpc, such as in the ratio between the labyrinth and the junctional zone. 189

Because deregulated imprinted genes expression is known to alter glycogen trophoblasts location, we 190 counted the number of vacuolated foci, graded the PAS staining, specific of glycoproteins, and 191 measured the surface of PAS area in the labyrinth. While in hemizygous TgSTOX13 placentas, no 192 significant difference was detected as compared to measures in wild-type placentas, in hemizygous 193 TgSTOX42 placentas, significant diminution of vacuolated foci in the labyrinth as well as PAS grade, 194 and PAS area (Figures 4B-D) was consistently observed. 195

Litter size is affected by the transgene

Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression 9 Litter size appeared linked to the genotypes of the males used in the crosses (Supplemental 197 Figure 2, p=0.0016 by one-factor ANOVA), with 8.4 (± 2.0) pups on average in WT crosses. In 198 crosses with TgSTOX13 males, the litter size was reduced to 6.9 (± 3.4, p = 0.048). Consistent with 199 the higher transgene expression in TgSTOX42, the litter size was reduced to 6.0 (± 2.6, p = 0.001) in 200 crosses involving a TgSTOX42 male. When aspirin was given to the females during gestation, litter 201 size was 7.8 (± 2.4) and 9. reproduce in extenso and without further manipulation the major features of preeclampsia: proteinuria 210 and hypertension. A peculiarity of the present mouse model of preeclampsia is the intensity of the 211 gestational hypertension, which reaches values corresponding to severe human preeclampsia at the end 212 of gestation. The lower difference in blood pressure observed under anesthesia compared to vigil 213 animal may presumably be explained by a relaxing effect of the anesthesia, moderating as well the 214 blood pressure level. We also observed an elevation of both sFlt1 and sEng, key anti-angiogenic 215 factors of preeclampsia. The mice also harbored PE-specific kidney alterations. 216

In summary, a unique transcription factor (STOX1) expressed in the foeto-placental unit is 217 sufficient to trigger preeclampsia-like symptoms in WT females, in a dose-dependent manner 218 (illustrated by the more or less severe phenotype observed in crosses involving TgSTOX13 or 219 TgSTOX42 males). While the involvement of STOX1 in human preeclampsia was debated after its 220 discovery [START_REF] Iglesias-Platas | STOX1 is not imprinted and is not likely to be 347 involved in preeclampsia[END_REF] , the present study confirms a growing corpus of evidence indicating an actual contribution 221 of STOX1 to the preeclamptic phenotype. 222 Rodent models of preeclampsia have been considered very important for studying 223 preeclampsia pathophysiological mechanisms. Various models of reduced uterine perfusion were 224 generated by inducing placental ischaemia, administration of angiotensin antagonists, pro-225 inflammatory drugs or vasoconstrictive agents, or by using the BPH/5 hypertensive mouse strain 21 , 226 and crosses between the CBA X DBA strains [START_REF] Ahmed | A new mouse model to explore therapies 352 for preeclampsia[END_REF] . Gene manipulations have also been used such as gene 227 invalidation experiments for p57Kip2 , Comt, or Corin [START_REF] Kanayama | Deficiency in p57Kip2 expression induces preeclampsia-like symptoms in mice[END_REF][START_REF] Kanasaki | Deficiency in catechol-O-methyltransferase 358 and 2-methoxyoestradiol is associated with pre-eclampsia[END_REF] , additive transgenesis of anti-angiogenic 228 factors such as s-flt1 [START_REF] Kumasawa | Pravastatin induces placental growth factor (PGF) and ameliorates 361 preeclampsia in a mouse model[END_REF] , or transgenesis leading to manipulating the renin/angiotensin system [START_REF] Falcao | Mice 363 overexpressing both human angiotensinogen and human renin as a model of superimposed 364 preeclampsia on chronic hypertension[END_REF] . In the 229 handful of existing mouse models of preeclampsia, gestational hypertension is mild [START_REF] Balta | Reduced 366 uterine perfusion pressure model is not successful to mimic severe preeclampsia[END_REF] , probably far 230 below the one of our model and far below what is observed in severe preeclampsia in women. 231 Moreover, we observed that the principal maternal symptom, gestational hypertension, appears very 232 early, from 3.5 days post coïtum. In humans, it has been shown that preeclampsia can exceptionally Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression 11 occur very early with a complete phenotypic profile before 15 weeks of gestation [START_REF] Imasawa | A case of "pure" preeclampsia with nephrotic syndrome before 370 15 weeks of gestation in a patient whose renal biopsy showed glomerular capillary 371 endotheliosis[END_REF] while in general 234 the symptoms are reported to appear from mid gestation (21-22 weeks) at the earliest. The occurrence 235 of the maternal hypertension at the very beginning of gestation in our model suggests that soluble 236 substances produced by the blastocyst, the embryo or even from the ejaculate may initiate the maternal 237 preeclamptic phenotype. It is interesting to compare hypertension curves obtained here with those of 238 most studies (such as 24 , 25 ) that show a raise during the last 5 days of gestation or so. This increase is 239 also visible in our model, but is superimposed on a highly elevated blood pressure triggered by 240 gestation. In sum our model cumulates presumably the effects of an early message from the embryo-241 foeto-placental unit to the mother systems controlling blood pressure (endothelial system and kidney), 242 and the effect of an increased placental/foetal demand occurring at the end of gestation, when the 243 rhythm of weight gain is the highest for the foetuses, this effect being the one reproduced in most 244 mouse models of preeclampsia. 245 Under anesthesia the difference in blood pressure measurement between the control pregnant 246 mice and the one crossed with transgenic males was present, albeit less pronounced than on vigil 247 animals. This is completely consistent with recent human data showing that under anesthesia, there is 248 a steep drop in maternal blood pressure but much more elevated in preeclamptic women than 249 normotensive pregnant women (-10% in normotensive versus -30% in preeclamptic women) [START_REF] Vricella | Epidural-associated hypotension is more 327 common among severely preeclamptic patients in labor[END_REF] . This 250 suggests that blood pressure variations in our model are very close to what is observed in human 251

clinics. 252

To the best of our knowledge, we demonstrate here for the first time in a mouse model, that 253 aspirin treatment at low doses administered in drinking water from the beginning of gestation is 254 efficient for preventing the maternal syndrome. In humans, it appears that a major issue is the timing 255 of aspirin administration: after 16 weeks of gestation it has no effect at all [START_REF] Bujold | 373 Prevention of preeclampsia and intrauterine growth restriction with aspirin started in early 374 pregnancy: a meta-analysis[END_REF] . A consensus tends 256 nevertheless to emerge, with the idea that aspirin is quite efficient when administered early (before the 257 end of the first trimester of pregnancy) [START_REF] Bujold | 373 Prevention of preeclampsia and intrauterine growth restriction with aspirin started in early 374 pregnancy: a meta-analysis[END_REF][START_REF] Beaufils | Prevention of pre-eclampsia by early antiplatelet 376 therapy[END_REF][START_REF] Wallenburg | Low-dose aspirin prevents 378 pregnancy-induced hypertension and pre-eclampsia in angiotensin-sensitive primigravidae[END_REF][START_REF] Bakhti | Prevention of gravidic endothelial hypertension by aspirin treatment 381 administered from the 8th week of gestation[END_REF][START_REF] Roberge | Early administration of low dose aspirin for the prevention of severe and mild 384 preeclampsia: a systematic review and meta-analysis[END_REF] . Aspirin at low doses may change the hemodynamic 258 properties of the blood thus preventing blood clotting, an issue that is sometimes associated with 259 Disseminated Intravascular Coagulation in severe preeclampsia with HELLP syndrome [START_REF] Mercer | Clinical characteristics and outcome of twin 386 gestation complicated by preterm premature rupture of the membranes[END_REF] .

Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression 12 Unexpectedly, mild anomalies of mature placenta (decrease of glycogen trophoblasts surface) 261 was only observed in crosses with TgSTOX42 males. Although we did not perform earlier histological 262 examination of developing placenta, focusing on spiraled arteries invasion for instance, the 263 observation that hypertension occurs before the formation of a placenta indicates that the mechanisms 264 underlying the observed phenotype is not only due to defaults in placentation. This opens the 265 possibility that different causes of preeclampsia may explain the grade of severity of preeclampsia 266 symptoms in Humans as well as the early or late onset of the disease. 267 Imprinted genes and among them the paradigmatic Igf2 are known to have a crucial role in 268 placental and foetal growth [START_REF] Constancia | Placental-specific IGF-II is a major modulator of 390 placental and fetal growth[END_REF] . In particular, the KO of Igf2 leads to non-development of glycogen 269 cells [START_REF] Lopez | Insulin-like growth factor II affects 392 the appearance and glycogen content of glycogen cells in the murine placenta[END_REF] . The observed defaults in glycogen trophoblasts in hemizygous TgSTOX42 placenta may be 270 related to the fact that STOX1 is an imprinted gene in humans and could thus regulate the other 271 members of the imprinted gene networks [START_REF] Varrault | Zac1 regulates an imprinted gene network 396 critically involved in the control of embryonic growth[END_REF] . 272

Perspectives 273

The mouse model presented here can help the understanding severe preeclampsia, for which 274 until now, very few experimental alternatives to the direct study of the patients are available. These 275 mice provide the community working on preeclampsia with a tool applicable for cognitive 276 (understanding preeclampsia pathogenesis) as well as applied (drug screening) usage, and even 277 potentially, deciphering how these drugs act. 278

Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression females crossed with transgenic animals, while those from females crossed with WT males were 436 completely free of this labeling. TgSTOX42 kidneys were overall more strongly labeled. Under 437 aspirin treatment (+ASP), the intensity of the labeling was dramatically reduced. 438

6 121 1 .

 61 Dunnett T-tests for comparison with the controls. The StatistXL add-in of Excel™ was systematically 118 used. P-values below 0.05 were considered significant. 119Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression Results Generation of STOX1 transgenic mice 122 Three independent transgenic lines (called TgSTOX13, TgSTOX34 and TgSTOX42) were 123 generated by introducing the human STOX1A (complete isoform) under the control of a ubiquitously 124 expressed promoter (the cytomegalovirus -CMV-promoter). TgSTOX13 and TgSTOX42 strains 125 contain a unique copy that was transmitted in a Mendelian fashion. As the expression level of 126 TgSTOX34 was very similar to that of TgSTOX13, we focused our attention on TgSTOX13 and 127 TgSTOX42 strains. 128

  TgSTOX13 (x2.0, p=0.04) and TgSTOX42 males (x2.3, p=0.01), compared to crosses with WT males. 165 3. Wild-type females carrying transgenic foetuses have kidney anomalies that are 166 characteristic of preeclampsia 167 In the kidneys of females crossed with TgSTOX13 mice, three glomerular changes were 168 observed: first, a glomerular tuft swelling with expansion of the capillary wall, up to the point that the 169 tuft could occasionally make contact with the Bowman's capsule; second, we observed, a slight Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression

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  3 (± 3.1) in crosses involving TgSTOX13 males TgSTOX42 males, 202 respectively, and in the later case, the difference was significantly different from non treated crosses 203 (p=0.022), showing that the drug was able to correct the 'reduced litter size' phenotype. 204 205 206 Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression 10 Discussion 208 Herein, we show that mice overexpressing STOX1 in the embryo-foeto-placental unit 209

None declared 291 410 Figure Legends 411 Figure 1 :

 2914104111 Figure Legends

Figure 2 :

 2 Figure 2: Increased plasmatic sFlt1 and sEng concentrations in WT females crossed with 424 STOX1 transgenic males 425

Figure 3 :

 3 Figure 3: Histological kidney alterations in WT females crossed with transgenic males at 16.5 430 days post coïtum (dpc). Renal histology of WT females crossed with WT, TgSTOX13 and 431TgSTOX42 males (HES coloration, scale bar = 100 µm); A) The glomerular size and the number of 432 cells were significantly augmented in the glomeruli of the mothers (WT) when the foetuses/placentas 433 were transgenic. We also observed a reduction of the endothelial fenestrae (** for p < 0.01, see text). 434 B) Anti-fibrin immunohistochemistry revealed a strong labeling (in brown) of the glomeruli from 435

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 18

Figure 4 :

 4 Figure 4: Reduced vacuolated foci, PAS staining, and PAS area in hemizygous TgSTOX13 439 placentas at 16.5 dpc. Indexes of glycogen trophoblast mislocation were measured in the labyrinthic 440 zone using vacuolated aspects of the foci under HE coloration and PAS staining (A, see Methods). 441 Several placentas from different crosses were used: 6 placentas from 3 different crosses for ♀WT x 442 ♂WT, ♀ WT x ♂ ht TgSTOX13; and ♀ WT x ♂ ht TgSTOX42. Consistently, numeration of 443 vacuolated foci (B), PAS grade (C) and PAS area (D) were significantly decreased in the labyrinth in 444 hemizygous TgSTOX42 placentas. 445

Table 1 447 448 Expression of the endogenous Stox1 gene and STOX1 transgene in mice in various tissues

 1 arbitrary units, the kidney expression in TgSTOX13 mice was set to 1 and served as a reference for 449 the expression of the transgene. The values of the endogenous are corrected by the Ct values of the 450 reporter gene (SDHA), giving a rough indication of the relative expression of the endogenous 451 compared to the transgene. 452

		placenta	kidney	Liver	brain	testis	spermatozoa
	WTxWT (endogenous
	Stox1, arbitrary	3000 ± 57 178 ± 4.3 92 ± 2.1 1646 ± 96	882 ± 19	358 ± 14
	units)
	TgSTOX13 *	2400 ± 90 1.0 ± 0.05 4.2 ± 0.15 1.9 ± 0.04 1646 ± 140	66 ± 4,7
	TgSTOX42 *	38088 ± 5540 12 ± 4.6 38.2 ± 11 1384 ± 100 7304 ± 2066 3755 ± 1195

*

increase in the glomerular cellularity (42.3 ± 10.1 in TgSTOX13 and 48.1 ± 11.9 in TgSTOX42 versus

Comment citer ce document : Doridot, L., Passet, B., Rigourd, V., Méharts, C., Barbaux, S., Ducat, A., Mondon, F., Vilotte, J. L., Castille, J., Breuiller-Fouché, M., Daniel, N., Le Provost, F., Bauchet, A.-L., Baudrie, V., Hertig, A., Buffat, C., Simeoni, U., Germain, G., Vilotte, J. L., Vaiman, D. (Auteur de correspondance) (2013). Preeclampsia-like symptoms induced in mice by feto-placental expression


E Attard
email: attard@hotmail.com

H Yang

A.-M Delort

P Amato

U Pöschl

C Glaux

T Koop

C E Morris
email: cindy.morris@avignon.inra.fr

Effects of atmospheric conditions on ice nucleation activity of Pseudomonas

Although ice nuclei from bacterial origin are known to be efficient at the highest temperatures known for ice catalysts, quantitative data are still needed to assess their role in cloud processes. Here we studied the effects of three typical cloud conditions (i) acidic pH (ii) NO 2 and O 3 exposure and (iii) UV-A exposure on the ice nucleation activity (INA) of four Pseudomonas strains. Three of the Pseudomonas syringae strains were isolated from cloud water and the phyllosphere and Pseudomonas fluorescens strain CGina-01 was isolated from Antarctic glacier ice melt. Among the three conditions tested, acidic pH caused the most significant effects on INA likely due to denaturation of the ice nucleation protein complex. Exposure to NO 2 and O 3 gases had no significant or only weak effects on the INA of two P. syringae strains whereas the INA of P. fluorescens CGina-01 was significantly affected. The INA of the third P. syringae strain showed variable responses to NO 2 and O 3 exposure. These differences in the INA of different Pseudomonas suggest that the response to atmospheric conditions could be strain-specific. After UV-A exposure, a substantial loss of viability of all four strains was observed whereas their INA decreased only slightly. This corroborates the notion that under certain conditions dead bacterial cells can maintain their INA. Overall, the negative effects of the three environmental factors on INA were more significant at the warmer temperatures. Our results suggest that in clouds where temperatures are near 0 • C, the importance of bacterial ice nucleation in precipitation processes could be reduced by some environmental factors.

Introduction

In clouds, ice formation at temperatures above about -38 • C occurs via heterogeneous ice nucleation initiated by ice nuclei (IN). Without these IN, water droplets in clouds would remain liquid until -38 • C, the temperature of spontaneous freezing initiated by homogeneous ice nucleation. A large diversity of particles act as IN in the atmosphere: mineral dust, metallic particles, soot, and biological particles (e.g. [START_REF] Demott | Measurements of the concentration and composition of nuclei for cirrus formation[END_REF]Möhler et al., 2007;[START_REF] Andreae | Aerosol-cloud-precipitation interactions. Part 1. The nature and sources of cloud-active aerosols[END_REF]. Among the biological particles, some bacteria are known as very efficient IN and notably some strains of Pseudomonas syringae which initiate freezing at temperatures as high as -2 • C [START_REF] Orser | Cloning and Expression of Bacterial Ice Nucleation Genes in Escherichia coli[END_REF]. The ice nucleation activity (INA) of bacteria is due to a specific protein complex on the bacterial outer membrane that permits efficient arrangement of water molecules into ice embryos. This arrangement is orchestrated by the INA protein which is composed of a repeated octapeptide [START_REF] Gurian-Sherman | Bacterial ice nucleation: significance and molecular basis[END_REF]. Aggregation of several ice nucleation-active protein modules leads to the formation of a protein complex whose size determines the level of INA: the larger the protein complex the higher the sub-zero temperature at which freezing is induced (Kozloff et al., 1991a;[START_REF] Turner | Three separate classes of bacterial ice nucleation structures[END_REF]. INA depends on the protein complex being held intact in the outer membrane of the bacteria. This condition is generally met by living cells, but dead cells can also have a high level of INA. This is the case for the commercial adjuvant for production Published by Copernicus Publications on behalf of the European Geosciences Union.

of artificial snow, Snomax , a preparation of freeze-dried irradiated cells of P. syringae.

Ice nucleation active particles of biological origin have been detected in numerous environments [START_REF] Bowers | Characterization of Airborne Microbial Communities at a High-Elevation Site and Their Potential To Act as Atmospheric Ice Nuclei[END_REF]Christner et al., 2008a;Christner et al., 2008b). INA bacteria have also been isolated from a range of substrates including plants [START_REF] Gross | Distribution, population dynamics, and characteristics of ice nucleation-active bacteria in deciduous fruit tree orchards[END_REF][START_REF] Kaneda | Seasonal population changes and characterization of ice-nucleating bacteria in farm fields of central Alberta[END_REF], aerosols, precipitation [START_REF] Lindemann | Plants as sources of airborne bacteria, including ice nucleation-active bacteria[END_REF][START_REF] Morris | The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle[END_REF] and from fresh water [START_REF] Morris | The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle[END_REF][START_REF] Morris | Inferring the evolutionary history of the plant pathogen Pseudomonas syringae from its biogeography in headwaters of rivers in North America, Europe, and New Zealand[END_REF]. Microbial species known to be ice nucleation active have been isolated from clouds [START_REF] Sands | The association between bacteria and rain and possible resultant meteorological implications[END_REF][START_REF] Amato | Microorganisms isolated from the water phase of tropospheric clouds at the Puy de Dôme: major groups and growth abilities at low temperatures[END_REF] and their ice nucleation activity was reported in one case [START_REF] Sands | The association between bacteria and rain and possible resultant meteorological implications[END_REF].

In clouds, there are particular environmental conditions such as light intensity and chemistry that could affect bacterial INA. However, quantitative data on such effects are lacking even though they could have considerable consequences for estimations of the impact of biological ice nucleation on cloud processes. Most of the data available on the effects of environmental conditions on bacterial INA are focused on (i) determining ice nucleation protein characteristics and structure (e.g. [START_REF] Turner | Three separate classes of bacterial ice nucleation structures[END_REF]Kozloff et al., 1991a, b), (ii) methods to increase ice nucleation active biomass and efficiency for industrial applications in the food industry and in snow making (e.g. [START_REF] Nemecek-Marshall | High-level expression of ice nuclei in a Pseudomonas syringae strain is induced by nutrient limitation and low temperature[END_REF][START_REF] Blondeaux | Influence of water activity on the ice-nucleating activity of Pseudomonas syringae[END_REF] and (iii) limiting frost damage to plants [START_REF] Anderson | The effects of streptomycin, desiccation, and UV radiation on ice nucleation by Pseudomonas viridiflava[END_REF][START_REF] Gurian-Sherman | Bacterial ice nucleation: significance and molecular basis[END_REF][START_REF] Kawahara | Identification of a compound in Chamaecyparis taiwanensis Inhibiting the Ice-Nucleating Activity of Pseudomonas fluorescens KUIN-1[END_REF]. To our knowledge only the study by [START_REF] O'brien | Effect of Plant Species and Environmental Conditions on Ice Nucleation Activity of Pseudomonas syringae on Leaves[END_REF] deviates from this tendency by analyzing the response of bacterial INA submitted to environmental conditions of the natural habitats of these bacteria. These authors observed that INA is sensitive to factors encountered on the plant surface and in particular those related to host plant genotype, light intensity and ambient humidity.

For non-biological ice nucleation particles, ample data are available about the influence of environmental conditions on their activity. For example, several studies have focused on the effects of sulfuric acid coating [START_REF] Archuleta | Ice nucleation by surrogates for atmospheric mineral dust and mineral dust/sulfate particles at cirrus temperatures[END_REF][START_REF] Knopf | Heterogeneous nucleation of ice on surrogates of mineral dust[END_REF][START_REF] Eastwood | Effects of sulfuric acid and ammonium sulfate coatings on the ice nucleation properties of kaolinite particles[END_REF], ammonium sulfate coating [START_REF] Eastwood | Effects of sulfuric acid and ammonium sulfate coatings on the ice nucleation properties of kaolinite particles[END_REF], organic coating [START_REF] Kanji | Ice formation via deposition nucleation on mineral dust and organics: dependence of onset relative humidity on total particulate surface area[END_REF][START_REF] Möhler | The effect of organic coating on the heterogeneous ice nucleation efficiency of mineral dust aerosols[END_REF], and ammonia gas [START_REF] Salam | Ice nucleation of ammonia gas exposed montmorillonite mineral dust particles, Atmos[END_REF] on the ice nucleation properties of particles. In certain studies [START_REF] Kanji | Ice formation via deposition nucleation on mineral dust and organics: dependence of onset relative humidity on total particulate surface area[END_REF][START_REF] Möhler | The effect of organic coating on the heterogeneous ice nucleation efficiency of mineral dust aerosols[END_REF] organic coating was found to deactivate ice nucleation, whereby a lower temperature is required to initiate ice formation. On the other hand, no common tendency was observed for other coatings and it was shown that effects of sulfuric acid coating depended on the IN material studied [START_REF] Archuleta | Ice nucleation by surrogates for atmospheric mineral dust and mineral dust/sulfate particles at cirrus temperatures[END_REF][START_REF] Knopf | Heterogeneous nucleation of ice on surrogates of mineral dust[END_REF][START_REF] Eastwood | Effects of sulfuric acid and ammonium sulfate coatings on the ice nucleation properties of kaolinite particles[END_REF]. Therefore, our study aimed at evaluating the effect of key conditions in clouds on bacterial INA. We examined the effects of the pH of cloud water, UV light and reactive gas exposure on the INA of bacteria isolated from various environments including clouds.

To evaluate the influence of pH on INA, we took into consideration the range of pH observed in cloud water. At the summit of the Puy de Dôme, for example, clouds of oceanic origin have a less acidic pH (pH 5.0 to 6.5) than clouds from the north that are influenced by anthropogenic activities (pH 3.0 to 4.5) (database available at http: //wwwobs.univ-bpclermont.fr/SO/beam/data.php; [START_REF] Marinoni | Cloud chemistry at the Puy de Dôme: variability and relationships with environmental factors[END_REF]. We hypothesized that acidification of cloud water can decrease the efficiency of bacterial INA probably via the denaturation of the tertiary structure of the ice nucleation protein and/or by an impact on bacterial metabolism.

To evaluate the role of important atmospheric chemical reactions on INA, bacterial strains were exposed to reactive gases. In heavily polluted urban areas there are high levels of NO 2 and O 3 in the atmosphere. It is known now that proteins can be subjected to oxidation and nitration when exposed to the gas mixtures of NO 2 , O 3 and water vapor [START_REF] Franze | Protein nitration by polluted air[END_REF][START_REF] Shiraiwa | The role of longlived reactive oxygen intermediates in the reaction of ozone with aerosol particles[END_REF][START_REF] Shiraiwa | Multiphase chemical kinetics of the nitration of aerosolized protein by ozone and nitrogen dioxide[END_REF]. In particular, tyrosine, an aromatic amino acid residue in proteins, can be efficiently nitrated [START_REF] Walcher | Liquid-and gas-phase nitration of bovine serum albumin studied by LC-MS and LC-MS/MS using monolithic columns[END_REF][START_REF] Yang | Quantification of nitrotyrosine in nitrated proteins[END_REF][START_REF] Zhang | Analysis of nitrated proteins and tryptic peptides by HPLC-chip-MS/MS: site-specific quantification, nitration degree, and reactivity of tyrosine residues[END_REF]. This amino acid residue is part of the water binding site of the ice nucleation protein conferring the tertiary structure with β-sheet folding at the glycinetyrosine-glycine site [START_REF] Gazit | Global analysis of tandem aromatic octapeptide repeats: The significance of aromatic-glycine motif[END_REF]. In this way the nitration reaction may change the structure of this amino acid that is involved in the tertiary structure of the protein thereby leading to changes in its water-binding capacity and hence its INA efficiency. We exposed bacteria to synthetic gas mixtures of NO 2 and O 3 at relevant atmospheric concentrations and examined the impact on INA.

To evaluate the effect of UV light, bacterial strains were exposed to UV-A, the most important source of photons in clouds which can cause intense oxidative damage to a range of molecules in bacterial cells [START_REF] Pourzand | Apoptosis, the role of oxidative stress and the example of solar UV radiation[END_REF]. We hypothesized that UV-A could affect INA directly via denaturation of the ice nucleation protein exposed on the outer cell membrane or indirectly due to cell death.

Materials and methods

Bacterial strains

Bacterial strains used in this study originated from three different environments. Strains 13b-2 and 32b-74 of P. syringae were isolated from cloud water collected at the Puy de Dôme summit (France) in July 2004 and November 2009, respectively. Sampling conditions and isolation methods have been described by [START_REF] Amato | Microbial population in cloud water at the Puy de Dôme: implications for the chemistry of clouds[END_REF]Vaïtinlingom et al. (2012). The 16S rRNA partial gene sequences of strains 13b-2 and 32b-74 have been submitted to the GenBank database (accession numbers DQ512785 and HQ256872). Strain P. syringae CC0242 was originally isolated from diseased cantaloupe leaves as described for closely related strains from the same origin [START_REF] Morris | The relationship of host range, physiology, and genotype to virulence on cantaloupe in Pseudomonas syringae from cantaloupe blight epidemics in France[END_REF]. Strain CGina-01of P. fluorescens was isolated from ice melt, provided by C. Foreman (Montana State University), from Cotton Glacier in Antarctica. Snomax is a commercial preparation containing a freeze-dried and irradiated preparation of a strain of P. syringae (Johnson Controls Inc, Milwaukee, USA). Before ice nucleation assays (described below), bacterial strains were cultured for 3 days at 17 • C on plates of King's medium B (KB) [START_REF] King | Two simple media for the demonstration of pyocyanin and fluorescein[END_REF]. Bacterial suspensions were prepared with the newly cultivated bacteria in sterile distilled water to obtain 5 × 10 8 -5 × 10 9 cells mL -1 . The suspensions were incubated at 4 • C for at least 1 hour to allow full expression of the ice nucleation protein.

Bacterial ice nucleation activity

Bacterial INA was measured by an immersion freezing test [START_REF] Vali | Quantitative evaluation of experimental results on the heterogenous freezing nucleation of supercooled liquids[END_REF]. Thirty-two droplets of 20 µl of the initial suspension and then 32 droplets of 10 fold serial dilutions of the suspension (from 5 × 10 8 to 5 × 10 3 cells mL -1 ) were distributed on a sterile aluminum plate and floated on a cooling bath (model F34-ED Julabo, Colmar, France). This serial dilution was performed to allow the calculations described below such that for at least one dilution series, some drops (at least 1) remained unfrozen.

The initial temperature was -2 • C and then reduced at 1 • C intervals until -10 • C. At each temperature step, the droplets were incubated for 8 min and at the end of each incubation step the number of frozen droplets was counted visually. The cumulative ice nuclei concentration per bacterial cell at each temperature was calculated according to the method described by [START_REF] Vali | Quantitative evaluation of experimental results on the heterogenous freezing nucleation of supercooled liquids[END_REF] with the following equation: c(T ) = [ln(N 0 ) -ln(N 0 -N x (T ))]/A where c(T ) is the number of ice nuclei per bacterial cell active at temperature T , N 0 is the number of droplets tested, N x is the number of frozen droplets at a given temperature T , and A is the number of bacteria per drop.

Therefore, for each bacterial strain, the level of the INA was determined by (i) the number of ice nuclei per cell and (ii) the highest temperature at which freezing was first observed.

To allow comparison of the INA of bacteria and the mineral compound montmorillonite, we calculated the ice nuclei concentration per surface area, assuming a bacterial surface of 5 µm 2 (considering bacteria as a cylinder of 1 µm in length and 0.5 µm of radius), and with 30 m 2 g -1 for montmorillonite [START_REF] Mortazavi | Ice nucleation activity of bacteria isolated from snow compared with organic and inorganic substrates[END_REF]. We chose to present the comparison between bacteria and this mineral IN because the publication of [START_REF] Mortazavi | Ice nucleation activity of bacteria isolated from snow compared with organic and inorganic substrates[END_REF] is, to our knowledge, the only study which has applied the immersion freezing assays to mineral compounds. Six replicates were used to calculate the ice nucleation spectra presented in Figs. 1 and4 replicates for all the experiments concerning pH, NO 2 /O 3 , and UV exposure effects.

Exposure to acidic pH

For these experiments, instead of serially diluting in distilled water, the initial bacterial suspension was serially diluted in one of two acetate buffers. The buffer at pH 5.9 was prepared with 93 mL of sodium acetate at 0.2 M and 7 mL of acetic acid at 0.2 M. The buffer at pH 4.1 was prepared with 18 mL of sodium acetate at 0.2 M and 82 mL of acetic acid at 0.2 M. Since the original two buffers had different sodium cation concentration, NaCl was added to the pH 4.1 buffer in order to achieve about the same sodium ion concentration in both buffers.

At room temperature acetic acid is liquid and dissolves in water at all concentrations. The acetic acid/H 2 O system shows a eutectic temperature (ice/acetic acid) of approximately -26.6 • C at which point the solubility of acetic acid is about 59 wt %, corresponding to a molality of about 24 mol kg -1 [START_REF] Dahms | Nachträge und Bemerkungen zu der Arbeit über Gefrierpunkte binärer Gemenge[END_REF]. For comparison, the maximum concentration of acetic acid in buffer 2 was only about 0.15 mol kg -1 . The solubility limit of aqueous sodium acetate solutions is actually determined by the solubility curve of its trihydrate (NaAc•3H 2 O). At room temperature the solubility is about 5.7 mol kg -1 and at the eutectic temperature of -18 • C it is 3.7 mol kg -1 [START_REF] Green | The "melting-point" of hydrated sodium acetate: solubility curves[END_REF]. In contrast, the maximum concentration of sodium acetate in buffer 1 was only about 0.17 mol kg -1 . These calculations show that neither sodium acetate nor acetic acid could have precipitated from the buffer solutions in our experiments upon cooling. We also tested the freezing of buffer -without bacteriaadjusted to pH 4.1 and 5.9. For each pH, 40 drops were tested for freezing down to -10 • C under the same conditions used to determine the INA of bacterial suspensions. No drops froze under these conditions.

It is well established that solutes affect both homogeneous and heterogeneous ice nucleation through a reduction in the aqueous solution's water activity [START_REF] Koop | Water activity as the determinant for homogeneous ice nucleation in aqueous solutions[END_REF][START_REF] Zobrist | Heterogeneous Ice Nucleation in Aqueous Solutions: the Role of Water Activity[END_REF]. This "colligative" effect on heterogeneous ice nucleation is different from that due to chemical, morphological, or other changes in the IN surface (e.g. the protein complex) induced by the presence of solutes. In order to discriminate between changes in the intrinsic IN properties and colligative effects, we have corrected the data for the colligative effect using a published protocol for estimating its magnitude [START_REF] Koop | Parameterizations for ice nucleation in biological and atmospheric systems[END_REF].

First, we calculated the concentration of all ionic and non-ionic solute species introduced by adding the buffer stock solutions described above, also considering the relevant dissociation equilibrium of HAc (Ks = 1.7540 × 10 -5 ). Because the total molality of all solute species was less than 0.5 mol kg -1 in both cases, we assumed ideal behaviour resulting in a w values of 0.99378 and 0.99160 for the pH 5.9 and pH 4.1/NaCl buffers, respectively. The observed freezing behaviour at these water activities was then corrected for the presence of solutes to yield the predicted freezing behaviour in pure water (a w = 1), see Supplement A for details. For example, the cumulative number of IN per bacterium in a pH 5.9 buffer measured at -3 • C corresponds to the same cumulative number of IN per bacterium in water at -2.34 • C. All corrections were smaller than 1 • C. The difference between the measured cumulative number of IN per bacterium in droplets with buffer corrected for solute effects and that measured separately in pure water without buffer is then attributed to the effect of pH owing to changes induced in the ice nucleating protein complex at the different pH.

Exposure to NO 2 /O 3

An aliquot of 110 µL of the prepared bacterial suspension was introduced into a sterile syringe filter (cellulose acetate membrane, pore size 1.2 µm, 30 mm diameter, sterile, Whatman GmbH, Dassel, Germany). For each exposure experiment, 6 filters were prepared. They were then dried by a gentle stream of high purity N 2 for ∼1-2 h. Two filters were kept in a sealed zip-lock bag without exposure to any synthetic gases as control samples to check the effect of exposure to gases with or without NO 2 and O 3 . The other 4 filters were exposed to synthetic gas mixtures overnight (16-18 h). A schematic diagram of the experimental exposure setup is shown in Supplement B. The exposure setup was the same as described in [START_REF] Yang | Quantification of nitrotyrosine in nitrated proteins[END_REF] except that a gas filter system was added upstream of two parallel syringe filters to obtain a synthetic gas mixture free of O 3 and NO 2 . So for each exposure experiment we had 6 samples. Two were control samples without exposure to any synthetic gas. Two of them were exposed to gas mixtures of 108 ± 2.7 ppb (average value ± standard deviation) NO 2 and 110 ± 11.3 ppb O 3 at the relative humidity (RH) of ∼50 %, the other two were exposed to filtered synthetic gas mixtures (major compositions: O 2 and N 2 ) without O 3 and NO 2 at a slightly reduced RH. After exposure, the filters were immediately washed-off with 1.1 mL of sterile high purity water. The suspension obtained was subsequently tested for its ice nucleation activity. To an aliquot of 100 µL of the remaining suspension, 5 µL of a 37 % (v/v) formaldehyde solution was added to fix bacterial cells for counting by flow cytometry.

UV exposure

An aliquot of 30 mL of the prepared bacterial suspension was placed in a crystallizer (d = 15 cm, h = 10 cm) with a Pyrex glass lid (thickness 3.3mm, cut-off wavelength: 290 nm, transparent to UV-A). The crystallizer was surrounded by a plastic circle with 10 aeration tubes to avoid water vapour condensation. In the control experiment, the crystallizer was entirely covered with aluminum foil to avoid UV-A light. Both of the crystallizers were incubated at 17 • C, with gentle shaking (50 rpm) and with UV-A light mimicking the solar spectrum in the atmosphere (i.e. mainly UV-A from 315 to 400 nm, 33 J s -1 m -2 , with the lamp and solar spectra as described in [START_REF] Vaïtilingom | Atmospheric chemistry of carboxylic acids: microbial implication versus photochemistry[END_REF]). An aliquot of 1 mL of the incubated bacterial suspension was taken at 0 h, 4 h, 20 h and 42 h and the ice nucleation activity was measured.

Statistical analyses

One-way ANOVA and Tukey's tests were performed with the PAST software [START_REF] Hammer | PAST: paleontological statistics software package for education and data analysis[END_REF] to determine if there were significant differences in the ice nucleation results obtained from different treatments.

Results

Bacterial ice nucleation activity

The bacterial strains collected from cloud water at the Puy de Dôme summit, P. syringae 13b-2 and 32b-74 showed marked INA. Both were collected from clouds originating from the French west coast with pH values of 5.2 and 5.5, respectively. Under our experimental conditions P. syringae 32b-74 initiated freezing at -3 • C and produced 10 -0.75 IN per bacterium, i.e. 1 active ice nucleus per 5.7 cells at the coldest temperature tested, -10 • C (Fig. 1a). Strain 13b-2, initiated freezing at -5 • C and produced two orders of magnitude fewer ice nuclei than strain 32b-74 at -10 • C (10 -3.4 IN per bacterium, i.e. 1 active nucleus per 2.5 × 10 3 cells at -10 • C) (Fig. 1a). Strain CC0242 of P. syringae isolated from the phyllosphere, strain CGina-01 of P. fluorescens from glacial melt and the Snomax commercial product had freezing profiles intermediate to strains 32b-74 and 13b-2. Except for strain 32-b74, all these strains had less activity than that of strain Cit7 of P. syringae under the conditions used by [START_REF] Orser | Cloning and Expression of Bacterial Ice Nucleation Genes in Escherichia coli[END_REF] (Fig. 1b). However, all of these strains had several orders of magnitude higher ice nucleation activity on a surface area basis than mineral dust e.g. montmorillonite at the temperature range between -3 and -10 • C (Fig. 1b, based on previously reported ice nucleation spectra of montmorillonite by [START_REF] Mortazavi | Ice nucleation activity of bacteria isolated from snow compared with organic and inorganic substrates[END_REF]. This mineral dust had 10 3.1 ice nuclei per m 2 surface area active at -10 • C whereas strains 32b-74 and 13b-2 had 10 10.4 and 10 7.8 ice nuclei per m 2 surface area of cell, respectively (Fig. 1b).

Effects of acidic pH

Composition of pH buffers led to a reduction in ice nucleation temperature of around 0.7 • C for the pH 5.9 buffer and of around 0.9-1.0 • C for the pH 4.1 buffer (Supplement C). The effect was slightly temperature dependent (i.e. a larger effect at a lower temperature) (Supplement C). Taking into account water activity corrections, the acidic pH 4.1 strongly reduced the IN number compared to pH 5.9 for the temperature range tested for strains 32b-74, 13b-2, CGina-01 and for Snomax (Fig. 2). For P. syringae CC0242, IN number at pH 4.1 compared to pH 5.9 was not significantly different. Compared to pH 5.9, the temperature at which freezing began was decreased at pH 4.1 for all strains except P. syringae 32b-74 (Fig. 2).

Compared to distilled water, pH 5.9 led to a decrease in the IN number for P. syringae 32b-74, P. fluorescens CGina-01 at the highest temperatures, and for all temperatures for P. syringae CC0242 (Fig. 2). Compared to distilled water, pH 5.9 led to an increase in the IN number for Snomax (Fig. 2).

Effects of NO 2 /O 3 exposure

The number of viable cells of all strains was significantly reduced by gas exposure. Taking into account that dead cells could maintain an efficient ice nucleation protein, INA was calculated by dividing the number of IN by the number of viable cells counted at the beginning of the experiment. In order to check if cells were degraded by gas exposure, the total cell number was counted by flow cytometry. This confirmed that the total cell number of all strains was not significantly affected by gas exposure (data not shown).

In order to evaluate the effect of the exposure systems, comparisons of INA were made between bacteria exposed to synthetic air without NO 2 /O 3 and bacteria not introduced in the exposure system. After exposure to synthetic air without NO 2 and O 3 , the INA of strains 32b-74 and 13b-2 was not significantly different from that of bacteria (at the same concentration) not introduced into the exposure system (data not shown). For strains CC0242 and CGina-01, after exposure to synthetic air without NO 2 and O 3 , the initial temperature of freezing decreased by 1 • C and 2 • C, respectively (data not shown).

In order to evaluate the effect of NO 2 /O 3 exposure, comparisons of INA were made between bacteria exposed to synthetic gas mixtures with and without NO 2 /O 3 . Exposure to NO 2 /O 3 did not have a significant effect on the INA of strain 13b-2 (Fig. 3). For strain 32b-74, NO 2 /O 3 exposure caused a decrease of 1 • C of the temperature at which freezing initially occurred and a slight difference in IN number at -4 • C (Fig. 3). In contrast, the INA of CGina-01 was significantly affected by NO 2 /O 3 exposure with a decrease of 2 • C in the temperature at which freezing initially occurred (Fig. 3). For CC0242, two different patterns of response were observed between the four replicate trials. In two replicates, the initial temperature at which freezing occurred was not affected by NO 2 /O 3 exposure whereas it was markedly decreased by 4 • C in the other two replicates (Fig. 3).

Effects of UV-A exposure

The number of viable cells of strains 32b-74 and 13b-2 was significantly reduced after 42 h of UV-A exposure (Fig. 4). Viable cell number of P. syringae CC0242 began to decrease after 20 h of UV-A exposure. The number of cells of CGina-01 decreased significantly from the first sampling point i.e. after 4 h of UV-A exposure. Whereas the viable cell number of P. syringae 32b-74, 13b-2 and CC0242 decreased by a factor of 10 at the end of the UV exposure, the abundance of viable cells of P. fluorescens CGina-01 decreased by a factor of 27.

For all strains, UV-A exposure did not lead to any significant effect on INA at 4 h and 20 h of exposure (data not shown). At the end of the exposure, no effect of UV-A exposure on the temperature at which freezing initially occurred was observed in any of the strains tested (Supplement D). INA of 13b-2 was not affected by 42 h of UV-A exposure (Supplement D). Conversely, the INA of 32b-74, CC0242 and CGina-01 increased after 42 h of UV-A exposure compared to unexposed control samples. Here, INA after 42 h of exposure was calculated by dividing the number of IN by the number of viable cells counted after 42 h of exposure. However, to take into account that dead cells could maintain an efficient ice nucleation protein, INA after 42 h exposure was also calculated by dividing the number of IN by the number of viable cells counted at the beginning of the experiment (Fig. 5). Consistent with the previous calculation, the INA of 13b-2 was not significantly affected by UV-A (Fig. 5). However, the INA of 32b-74, CC0242 and CGina-01 was slightly reduced by UV-A exposure.

Discussion

There has been a recent focus of atmosphere modeling on evaluation of the impact of biological ice nuclei on glaciation processes leading to precipitation [START_REF] Hoose | How important is biological ice nucleation in clouds on a global scale?[END_REF]Sesartic et al., 2011). However this approach is limited by quantitative data on abundance and activity of biological ice nucleators in the atmosphere. Here we reported not only ice nucleation spectra of different bacterial strains but also their response to three environmental conditions typically encountered in clouds. Our results demonstrate that among these conditions, acidic pH has a strong effect on bacterial INA but UV-A and NO 2 /O 3 exposure have only little effects. Furthermore, the pH effects on INA are more evident at higher temperatures but by -8°or -10 • C the effects are weaker. In this work we demonstrated that acidic pH reduced the INA of all the four strains studied and more intensely above -8 • C. This effect of acidic pH on INA of Pseudomonas was previously studied by [START_REF] Kozloff | Ice nucleating activity of Pseudomonas syringae and Erwinia herbicola[END_REF] and [START_REF] Kawahara | Isolation and characterization of a novel ice-nucleating bacterium, Pseudomonas, which has stable activity in acidic solution[END_REF] whose results are consistent with ours. In addition, [START_REF] Turner | Three separate classes of bacterial ice nucleation structures[END_REF] reported that effects of acidic pH were more intense for ice nuclei active at temperatures near 0 • C. They observed that the number of bacterial ice nuclei active at -4 • C was severely reduced by acidic pH whereas those active at -9 • C were almost not affected. Similarly, at a colder temperature (-38 • C), no effects of acidic pH were observed on the IN properties of Snomax [START_REF] Chernoff | Effects of sulfate coatings on the ice nucleation properties of a biological ice nucleus and several types of minerals[END_REF]. As it has been proposed that ice nucleation efficiency is linked to the level of aggregation of the ice nucleation protein (Kozloff et al., 1991a) these results suggest that acidic pH acts via denaturation of the larger protein complexes. Indeed, bacteria expressing INA at the warmer temperatures carry larger protein complexes whereas at colder temperature smaller complexes are more abundant (Kozloff et al, 1991a). Then with acidic pH leading to denaturation of the protein complex, more effect will be observed on the larger complexes active at high temperatures and weaker effects will be observed on the smaller complexes active at cold temperatures. Furthermore, effects of acidic pH at the warmer temperatures were observed not only on the INA of living bacteria but also on the INA of the non-viable bacterial product Snomax , thereby supporting the hypothesis that acidic pH affects the INA of bacteria probably by directly changing the ice nucleation protein structure. However, the effects of acidic pH were less intense on Snomax than on living bacteria. This suggests that for living cells, acidic pH likely acts through a combination of direct changes of the ice nucleation protein structure and indirect effects via bacterial metabolism in reaction to acidic conditions.

12 -10 -9 -8 -7 -6 -5 -4 -3 -2 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 -10 -9 -8 -7 -6 -5 -4 -3 -2 -10 -9 -8 -7 -6 -5 -4 -3 -2 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 -10 -9 -8 -7 -6 -5 -4 -3 -
To more clearly specify the effects of pH on INA, we took into account the effect on water activity of the solutes used as buffer. Indeed, as shown previously [START_REF] Zobrist | Heterogeneous Ice Nucleation in Aqueous Solutions: the Role of Water Activity[END_REF][START_REF] Koop | Parameterizations for ice nucleation in biological and atmospheric systems[END_REF] a reduction in water activity is linked to a reduction in the INA of dusts and Snomax . All our data were corrected for water activity of the pH buffer but, when subjected to pH 5.9, the INA of some strains differed more or less from that in distilled water. For Snomax , a slight positive effect of this pH buffer on INA was observed. This increase is counterintuitive but could be linked to the complex nature of Snomax which is a lyophilized product composed of bacteria but also culture medium residues and different wastes which can interact in a complex and still unexplained way with the pH buffer.

In contrast to the strong effects of acidic pH on INA, NO 2 /O 3 exposure did not significantly influence the INA of strains P. syringae 32b-74 and 13b-2, which seem to indicate that the structure of the IN-inducing protein on the surface of the bacteria was not significantly changed by exposure to the reactive gases NO 2 and O 3 . For P. fluorescens a reduction in the temperature at which freezing began was observed. It should be noted that the INA of this strain was already reduced by exposure to the synthetic gases without O 3 and NO 2 . This fragility could account for the greater sensitivity of P. fluorescens to exposure to NO 2 and O 3 gases than of the P. syringae strains. The effect on strain CC0242 of P. syringae was more complicated than for the others strains: whereas the INA of two replicates was only slightly reduced, the INA of two others replicates was substantially affected by the reactive gas exposure. Variability among tests of INA was reported before. [START_REF] Mortazavi | Ice nucleation activity of bacteria isolated from snow compared with organic and inorganic substrates[END_REF] have suggested that it is likely due to the ability of the ice-nucleating protein to aggregate. Despite the well-controlled conditions of preparation of bacteria for testing, some unidentified parameters may lead to differences in aggregation and thus to variations of INA.

Concerning UV-A exposure, we observed a significant loss of viability for all strains and especially for strain CGina-01 of P. fluorescens. Strikingly, this significant loss of viability was not associated with a decrease in INA (calculated with cell number at the beginning of the experiment) further validating the fact that under certain conditions, non viable cells can maintain INA and especially when cell integrity is maintained (Kozloff et al., 1991a). However, it should be noted that certain UV wavelengths can be damaging for INA. As shown by [START_REF] Anderson | The effects of streptomycin, desiccation, and UV radiation on ice nucleation by Pseudomonas viridiflava[END_REF] the highly lethal UV-C (almost absent in the atmosphere) caused a total loss of viability and a large reduction in INA, contrary to our observation of the effects of UV-A exposure.

Conclusions

Our results make a contribution to efforts to elucidate the impact of biological ice nucleators on cloud glaciation, but overall they illustrate the need for more in-depth quantification of the abundance and activity of INA organisms in the atmosphere. Firstly, it is interesting to note that two strains used here were isolated from cloud water providing direct evidence of their presence and viability in this environment. Furthermore, not all the P. syringae strains isolated in clouds were active as IN contrary to what was observed in snow and rain samples [START_REF] Morris | The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle[END_REF]. This could suggest that INA is not necessarily involved in the incorporation of these bacteria in cloud water whereas it might be for incorporation into precipitation. Our results also illustrate that the ice nucleation activity of Pseudomonas is surprisingly robust in response to atmospheric conditions other than acidity. Hence the data presented here can contribute to efforts to model cloud glaciation processes in that they suggest that the efficiency of some of the biological catalysts of ice formation in clouds might be reduced in clouds acidified by anthropogenic emissions. Such information can be incorporated in regional models but might be difficult to consider in global models. Exposure to other pollutants such as NO 2 /O 3 seems to have a weaker effect. However, the differences in behaviour that we observed among strains and between the two species studied here suggest that the response to atmospheric conditions is rather strain-specific.

Acidic pH of atmospheric water droplets is a modern phenomenon due to the strong increase in emissions of SO 2 and NO x from the 19 th century, the beginning of the industrialization era [START_REF] Smith | Historical Sulfur Dioxide Emissions 1850-2000: Methods and Results[END_REF][START_REF] Dentener | Emissions of primary aerosol and precursor gases in the years 2000 and 1750 prescribed data-sets for Ae-roCom[END_REF][START_REF] Lamarque | Historical (1850-2000) gridded anthropogenic and biomass burning emissions of reactive gases and aerosols: methodology and application[END_REF][START_REF] Skeie | Anthropogenic radiative forcing time series from preindustrial times until 2010[END_REF]. If initiating cloud glaciation processes is part of an adaptive survival and dissemination strategy for air-borne bacteria such as P. syringae, then it would be expected that the INA of these bacteria would not be significantly deteriorated by common conditions encountered in the atmosphere -and that likewise there could be selective pressures that will lead to eventual adaptation of P. syringae and other IN bacteria to these emerging conditions. Therefore, unlike mineral nucleators, it is feasible that, with continued exposure to atmospheric pollutants, biological ice nucleators in the atmosphere would adapt to the changing atmospheric conditions -and in particular chemical conditions -leading to enhanced efficiency of their INA. The challenge for cloud modelling will be to integrate these dynamic parameters.

Figure 1 :

 1 Figure 1: Ice nucleation spectra of Pseudomonas strains in distilled water: P. syringae 32b-74 3

Fig. 1 .

 1 Fig. 1. Ice nucleation spectra of Pseudomonas strains in distilled water: P. syringae 32b-74 and 13b-2 •, P. syringae CC0242 , P. fluorescens CGina-01 , the commercial Snomax , P. syringae Cit7 (grey dotted line; data from Orser et al., 1985) and Montmorillonite (grey solid line, data from Mortazavi et al., 2008). A: log (cumulative IN per bacterium); B: log (cumulative IN per m 2 ). Errors bars indicate standard errors (n = 6).

Figure 2 :6Fig. 2 .

 22 Figure 2: Ice nucleation spectra of Pseudomonas strains submitted to different pH: pH=4.1 3

Figure 3 :

 3 Figure 3: Ice nucleation spectra of Pseudomonas strains exposed to synthetic gases with (solid

  11

Fig. 3 . 2 Figure 4 :

 324 Fig. 3. Ice nucleation spectra of Pseudomonas strains exposed to synthetic gases with (solid line) and without (dotted line) NO 2 and O 3 .The horizontal line corresponds to the lower limit of detectable activity depending on the initial bacterial concentration of each experiment. Symbols are as in Fig.1. Each symbol corresponds to the mean value of 4 replicates except for P. syringae CC0242 that was exposed to gases with NO 2 and O 3 , where symbols correspond to the mean value of 2 replicates. At each temperature, values associated with different letters are significantly different (p < 0.05) based on Tukey's test. ns: no significant treatment effect. Errors bars are standard errors (n = 4, except for P. syringae CC0242 where n = 2).

Fig. 4 .

 4 Fig. 4. Viable cell number (CFU) of the Pseudomonas strains after UV exposure (solid line) or darkness (dotted line). Symbols are as in Fig. 1. At each time point, values associated with an asterisk are significantly different (p < 0.05) based on Tukey's test. Errors bars are standard errors (n = 4).

Figure 5 :5

 5 Figure 5: Ice nucleation spectra of Pseudomonas strains submitted to 42 h of UV-A exposure

Fig. 5 .

 5 Fig. 5. Ice nucleation spectra of Pseudomonas strains submitted to 42 h of UV-A exposure (solid line) or darkness (dotted line). The horizontal line corresponds to the lowest value of detectable activity, which depends on the initial bacterial concentration of each experiment. Symbols are as in Fig. 1. INA was calculated by dividing the number of ice nuclei (IN) recorded after 42 h of UV-exposure by the viable cell number counted at the beginning of the experiment. At each temperature, values associated with different letters are significantly different at p < 0.05 and those associated with an asterisk are significantly different at p < 0.1 based on Tukey's test. ns: no significant treatment effect. Errors bars are standard errors (n = 4).

Atmos. Chem. Phys., 12, 10667-10677, 2012 www.atmos-chem-phys.net/12/10667/2012/

www.atmos-chem-phys.net/12/10667/2012/ Atmos. Chem. Phys., 12, 10667-10677, 2012

We thank X. Chi, J. Fröhlich, I. Müller, C. Pöhlker, C. Ruzene, for technical support. We thank C. Foreman (Montana State University) for providing the ice melt sample from which strain CGina-01 was isolated. Publication costs were covered by a grant from the US National Science Foundation Division of Atmospheric and Geospace Sciences to C.E.M. Edited by: A. Nenes www.atmos-chem-phys.net/12/10667/2012/ Atmos. Chem. Phys., 12, 10667-10677, 2012

Supplementary material related to this article is available online at: http://www.atmos-chem-phys.net/12/ 10667/2012/acp-12-10667-2012-supplement.pdf.

Acknowledgements. Postdoctoral funding for E. Attard was supplied by the Institut de Chimie de Clermont-Ferrand (France) thanks to CNRS and Blaise Pascal University (UBP) fellowships.

The experiment part with NO 2 /O 3 exposure was funded by the Max Planck Society (MPG). We acknowledge funding from the joint DFG-CNRS project Bioclouds for T. K. (under KO 2944/1-1), E. A. and A. M. D.