Aurélie Nakamura
email: aurelie.nakamura@iplesp.upmc.fr

Laura Pryor

Morgane Ballon

Sandrine Lioret

Barbara Heude

Marie-Aline Charles

Maria Melchior

El-Khoury Lesueur

Maternal education and offspring birthweight for gestational age: the mediating effect of smoking during pregnancy

Keywords: Birthweight, educational attainment, tobacco smoking, pregnancy, mediation analysis, health inequalities

Background Small for gestational age (SGA) birthweight, a risk factor of infant mortality and delayed child development, is associated with maternal educational attainment. Maternal tobacco smoking during pregnancy could contribute to this association. We aimed to quantify the contribution of maternal smoking during pregnancy to social inequalities in child birthweight for gestational age (GA).

Methods Data come from the French nation-wide ELFE cohort study, which included 17,155 singletons. Birthweights for GA were calculated using z-scores. Associations between maternal educational attainment, tobacco smoking during pregnancy and child birthweight for GA were ascertained using mediation analysis. Mediation analyses were also stratified by maternal pre-pregnancy body mass index.

Results

Low maternal educational attainment was associated with an increased odd of tobacco smoking during pregnancy (adjusted OR (ORa)=2.58 [95% CI 2.34, 2.84]) as well as a decrease in child birthweight for GA (RRa=0.94 [95% 0.91, 0.98]). Tobacco smoking during pregnancy was associated with a decrease in offspring birthweight for GA (RRa=0.73 [95% CI 0.70, 0.76]). Mediation analysis suggests that 39% of the effect of low maternal educational attainment on offspring birthweight for GA was mediated by smoking during pregnancy. A more important direct effect of maternal educational attainment on child birthweight for GA was observed among underweight women (RRa=0.82 [95%CI 0.72, 0.93]).

Conclusions

The relationship between maternal educational attainment and child birthweight for GA is strongly mediated by smoking during pregnancy. Reducing maternal smoking could lessen the occurrence of infant SGA and decrease socioeconomic inequalities in birthweight for GA.

Introduction

Approximately 5 to 15% of singletons born in Western countries were born small for gestational age (SGA), which is a risk factor for mortality, [START_REF] Ruiz | Mother's education and the risk of preterm and small for gestational age birth: a DRIVERS meta-analysis of 12 European cohorts[END_REF][START_REF] Ludvigsson | Small for gestational age and risk of childhood mortality: A Swedish population study[END_REF] delayed development and poor health throughout the lifecourse. [START_REF] Barker | Fetal nutrition and cardiovascular disease in adult life[END_REF] Risk factors for SGA birth include socioeconomic disadvantage, [START_REF] Blumenshine | Socioeconomic disparities in adverse birth outcomes: a systematic review[END_REF] possibly because of differences in health behaviors, [START_REF] Lanting | Clustering of Socioeconomic, Behavioural, and Neonatal Risk Factors for Infant Health in Pregnant Smokers[END_REF] health care access and health literacy across socioeconomic groups. [START_REF] Azria | Inégalités sociales en santé périnatale[END_REF] Women's socioeconomic position can be assessed by the highest level of education attained, considered one of the best predictors of pregnancy outcomes. [START_REF] Mortensen | Social inequality in fetal growth: a comparative study of Denmark, Finland, Norway and Sweden in the period 1981-2000[END_REF] One of the health behaviours most strongly associated with maternal educational attainment is tobacco smoking, [START_REF] Hiscock | Socioeconomic status and smoking: a review[END_REF] which is also an important risk factor for adverse perinatal outcomes. [START_REF] Pereira Pp Da | Maternal active smoking during pregnancy and low birth weight in the Americas: a systematic review and meta-analysis[END_REF] Prior research suggests that maternal smoking early in pregnancy contributes to social inequalities in SGA, [START_REF] Van Den Berg | Educational inequalities in perinatal outcomes: the mediating effect of smoking and environmental tobacco exposure[END_REF][START_REF] Huijbregts | Interrelations between maternal smoking during pregnancy, birth weight and sociodemographic factors in the prediction of early cognitive abilities. Infant and Child Development[END_REF][START_REF] Beard | Socioeconomic and maternal determinants of small-for-gestational age births: patterns of increasing disparity[END_REF] however its role throughout pregnancy has not been thoroughly documented. In addition, prior studies neither systematically accounted for important confounders nor applied rigorous methods to study mediation. One study based on multiple mediation analyses [START_REF] Ballon | Which modifiable prenatal factors mediate the relation between socio-economic position and a child's weight and length at birth? Maternal & child[END_REF] and one study based on path analysis 14 also suggested smoking during pregnancy could be a mediator in the relation between maternal educational attainment and SGA offsprings. In France, women's tobacco smoking rates are among the highest in Europe: approximately 30% of women of child bearing age are daily smokers, [START_REF]Smoking prevalence, females (% of adults) 2015[END_REF] and 17% use tobacco throughout pregnancy. [START_REF] Andler | Baromètre santé 2017. Consommations d'alcool et de tabac durant la grossesse[END_REF] Socioeconomically disadvantaged women have higher levels of smoking and are more likely to continue smoking through pregnancy than those who have more favourable backgrounds. [START_REF] El-Khoury | Smoking Trajectories during the Perinatal Period and Their Risk Factors: The Nationally Representative French ELFE (Etude Longitudinale Française Depuis l'Enfance) Birth Cohort Study[END_REF] At the same time, low maternal educational attainment is associated with a higher maternal body mass index (BMI), [START_REF] Sundquist | The influence of socioeconomic status, ethnicity and lifestyle on body mass index in a longitudinal study[END_REF] which could be protective against having a SGA offspring, [START_REF] Yu | Pre-pregnancy body mass index in relation to infant birth weight and offspring overweight/obesity: a systematic review and meta-analysis[END_REF] therefore maternal prepregnancy BMI needs to be accounted for when examining the contribution of tobacco smoking. [START_REF] Mortensen | The social gradient in birthweight at term: quantification of the mediating role of maternal smoking and body mass index[END_REF] In this study we quantify the mediating effect of tobacco smoking during pregnancy in the association between maternal educational attainment and offspring birthweight for gestational age (GA) in a large French nation-wide birth cohort study (ELFE).

Methods

Study design and participants

ELFE (Etude Longitudinale Française depuis l'Enfance) is a nationally-representative birth cohort that follows 18,275 children born in France in 2011. [START_REF] Charles | Cohort Profile: The French National cohort of children ELFE: birth to 5 years[END_REF] Children were recruited in 320 maternity wards using random sampling. To be included, children had to be singletons or twins born at 33 weeks of gestation or older. Mothers had to be at least 18 years of age and not planning to move outside of Metropolitan France in the three years following study inclusion. Mothers had to be able to give consent in French, English, Arabic or Turkish. Data were collected at birth via face-to-face interviews conducted by midwives and via selfreported questionnaires. The participation rate was of 51%. [START_REF] Charles | Cohort Profile: The French National cohort of children ELFE: birth to 5 years[END_REF] The present analysis included a sample of 17,155 singletons with complete data on birthweight, gestational age and sex.

Measures

Maternal educational attainment

Maternal educational attainment was categorized into 1) high (>high school degree) or 2) low (≤high school degree).

Tobacco smoking during pregnancy

Tobacco smoking during pregnancy was assessed retrospectively in face-to-face interviews conducted at the time of the child's birth: 'Did you smoke during your pregnancy, even if it was from time to time?' (yes/no).

Child birthweight for gestational age

The child's birthweight was extracted from medical records. Birthweights were normalized according to GA and sex (z-scores). [START_REF] Hertz-Picciotto | Comparisons of infant mortality using a percentilebased method of standardization for birthweight or gestational age[END_REF]

Covariates

Several factors potentially associated with either smoking during pregnancy [START_REF] El-Khoury | Smoking Trajectories during the Perinatal Period and Their Risk Factors: The Nationally Representative French ELFE (Etude Longitudinale Française Depuis l'Enfance) Birth Cohort Study[END_REF] or child birthweight [START_REF] De Bernabé | Risk factors for low birth weight: a review[END_REF] were examined, including demographic characteristics: maternal age at birth, nationality (French vs. other), parity (0 vs. ≥1), residential area (Paris region vs. other), relationship status (living with a partner: yes vs. no); maternal health and perinatal characteristics: body mass index (BMI) before pregnancy (in kg/m 2 ), attendance of at least one antenatal education class (yes vs. no), number of prenatal visits, severe psychological difficulties during pregnancy (yes vs. no), alcohol consumption during pregnancy (>once a month vs. ≤once a month), pregnancy complications (e.g. high blood pressure, gestational diabetes, bleeding, etc.: ≥1 vs. <1 complication), gestational weight gain (in kg), physical activity during pregnancy (based on the Pregnancy Physical Activity Questionnaire [START_REF] Chasan-Taber | Development and validation of a pregnancy physical activity questionnaire[END_REF] where the overall score, ranging from 0.2 to 1292.4, was categorized as lower vs. greater than the median value (158.9, mean=175.6 (88.9)) or missing: physically active vs. physically inactive vs. missing). We also controlled for maternity type (public vs. private) and child sex. Birthweight, GA, child sex and pregnancy complication information was obtained through medical records. All other variables were self-reported by face to face interview, except for physical activity during pregnancy that was reported by telephone interview. [START_REF] Charles | Cohort Profile: The French National cohort of children ELFE: birth to 5 years[END_REF]

Statistical analyses

First, bivariate analyses were conducted to test associations between 1) maternal educational attainment and tobacco smoking during pregnancy, 2) maternal educational attainment and offspring birthweight for GA and 3) maternal tobacco smoking during pregnancy and offspring birthweight for GA. Then, using multivariable linear regression models, we adjusted for covariates associated either with maternal educational attainment, tobacco smoking during pregnancy or offspring birthweight for GA in bivariate analyses.

Then, we analysed associations between maternal educational attainment, tobacco smoking during pregnancy and offspring birthweight for GA in a counterfactual mediation analysis model, as defined by VanderWeele and colleagues [START_REF] Vanderweele | Odds ratios for mediation analysis for a dichotomous outcome[END_REF] . In brief, this method allows the decomposition of the effects of maternal educational attainment on offspring birthweight for GA in a natural direct effect and an indirect effect passing through maternal tobacco smoking during pregnancy in a single model. Direct and indirect effects of maternal educational attainment and the proportion of the association with offspring birthweight mediated by tobacco smoking during pregnancy were estimated as division of the indirect effect on the total effect [START_REF] Vanderweele | Odds ratios for mediation analysis for a dichotomous outcome[END_REF] . Figure 1 illustrates the theoretical model in a diagram acyclic graph (DAG) of the hypothesized mediation mechanism. In additional analyses, we tested for an interaction between maternal educational attainment (exposure) and tobacco smoking during pregnancy (potential mediator). The mediation analysis was carried out on complete observations. Finally sensitivity analyses regarding robustness of the results to potential unmeasured or uncontrolled confounders were conducted by calculating E-values. The Evalue "represents minimum strength of association, on the risk ratio scale, that an unmeasured confounder would need to have with both the treatment and the outcome to fully explain away a specific treatment-outcome association, conditional on the measured covariates". [START_REF] Vanderweele | Sensitivity analysis in observational research: introducing the Evalue[END_REF] The closer to one the E-value is, the greater are chances that unmeasured confounders could bias our estimates. In our mediation model, unmeasured confounders could affect estimates of associations between 1) maternal education attainment and maternal tobacco smoking during pregnancy 2) maternal tobacco smoking and offspring birthweight for GA and 3) maternal education attainment and offspring birthweight for GA.

Thus, three E-values were calculated.

All analyses were conducted with SAS 9.4. (SAS Inst Cary NC. 2003) Mediation analyses were implemented using the SAS macro "%mediation" developed by Valeri and VanderWeele [START_REF] Valeri | Mediation analysis allowing for exposure-mediator interactions and causal interpretation: Theoretical assumptions and implementation with SAS and SPSS macros[END_REF] . E-values and 95% confidence intervals were calculated using the R package EValue. [START_REF] Mathur | Web site and R package for computing Evalues[END_REF] In supplementary analyses, to account for the possible moderating role of BMI in the relationship between mother's education level and child's birthweight, we repeated the mediation analyses stratifying by pre-pregnancy maternal BMI, categorized as 1) low (<18.5kg/m 2 ), 2) normal (≥18.5 and <25kg/m 2 ), 3) or overweight (≥25 and <30kg/m 2 ) and high (≥30kg/m 2 ).

Results

Characteristics of ELFE cohort study participants

Descriptive statistics of women included in the analysis are presented in Supplementary Table 1S. About 60% of mothers had high educational attainment. Twenty percent of mothers reported smoking tobacco during pregnancy. Children's birthweight was on average 3,335g (479 g). The percentage of missing data varied between 0 and 3%, except for physical activity during pregnancy (25% missing values). Compared to children included in the study, those who were excluded from the analyses because of missing values on covariates (n=2,737) were significantly more likely to be born to mothers who had low educational attainment (44% vs. 41%, P<0.001), but there were no differences in terms of pre-term births (5% vs. 4%, P=0.1), birthweight (3,330g (507g) vs. 3,335g (474g), P=0.6) and low birthweight for GA (9% vs. 8%, P=0.4) (data available upon request).

Mediation analysis

Women with low educational attainment had an increased risk of having a child with decreased birthweight for GA compared to those who had high educational attainment (crude RR=0.94 [95% CI 0.91, 0.97]). After adjustment for potential confounders (all covariates listed above), this association remained stable (RRa=0.94 [95% CI 0.91, 0.98]). Low educational attainment was also associated with an increased odd of tobacco smoking during pregnancy, even after adjustment for potential confounders (ORa=2.58 [95% CI 2.35, 2.84]). Tobacco smoking during pregnancy was associated with a decreased birthweight for GA, even after adjustment for potential confounders (RRa=0.73 [95% CI 0.70, 0.76) (Figure 2). Women's educational attainment was associated with tobacco smoking during pregnancy, which in turn was associated with offspring birthweight for GA, allowing for mediation analyses. Natural direct and indirect effects and proportion mediated of birthweight weight for GA, without interactions between educational attainment and tobacco smoking during pregnancy, are presented in Supplementary Figure 1S. We found a natural direct effect of educational attainment on offspring birthweight for GA (RR direct =0.94 [95% CI 0.91, 0.98]). We also observed a statistically significant adverse natural indirect effect of low educational attainment on offspring birthweight for GA via an increase in tobacco smoking during pregnancy (RR indirect =0.96 [95% CI 0.95, 0.96]). In additional analyses, we included an interaction between educational attainment and tobacco smoking during pregnancy in our models, yielding comparable direct and indirect risk ratios. The interaction term between educational attainment and tobacco smoking during pregnancy was not statistically significant (P= 0.6), thus we did not include it in further analyses. Overall, we estimated that 39% of the effect of maternal educational attainment on offspring birthweight for GA was mediated by maternal tobacco smoking during pregnancy.

Stratification by maternal pre-pregnancy BMI

The association between maternal educational attainment and offspring birthweight for GA was stronger in underweight women (RRa=0.82 [95% CI 0.72, 0.93]) than in women with normal or high BMI. The association between maternal tobacco smoking during pregnancy and infant birthweight for gestational age was stronger in overweight women (RRa=0.70 [95% CI 0.63, 0.77]) than in other groups. (Table 1)

Nevertheless, the indirect effect of maternal educational attainment on infant birthweight for GA, mediated by tobacco smoking during pregnancy, was consistent across all strata of maternal BMI. Thus, the proportion of the effect of maternal educational attainment mediated (PM) by tobacco smoking was lower in underweight women (PM=23%) than in women with normal BMI (PM=47%). (Figure 3)

Discussion

Principal findings

Studying a large national cohort of women and their children, we found that low maternal educational attainment predicts a 1.1 fold decrease in child birthweight for GA. Up to 39% of this association appears to be due to differences in maternal tobacco smoking during pregnancy. Importantly, tobacco smoking plays a lesser role with regard to social inequalities in birthweight for GA in children of women who are underweight and who might have other specific risk factors. To the extent that birthweight predicts a number of health outcomes later in life (e.g. psychological and cognitive development, overweight/obesity, cardiovascular disease), [START_REF] Melchior | Maternal tobacco smoking in pregnancy and children's socio-emotional development at age 5: The EDEN mother-child birth cohort study[END_REF][START_REF] Pettersson | Birth weight as an independent predictor of ADHD symptoms: a within-twin pair analysis[END_REF] exposure to tobacco smoking in pregnancy could play an important role in socioeconomic disparities in health throughout the life course.

Our findings are in line with prior studies conducted in other settings and using other methods. In a study conducted in the Netherlands, van den Berg and colleagues [START_REF] Van Den Berg | Educational inequalities in perinatal outcomes: the mediating effect of smoking and environmental tobacco exposure[END_REF][START_REF] Van Den Berg | Smoking overrules many other risk factors for small for gestational age birth in less educated mothers[END_REF] found that tobacco smoking during pregnancy mediated both the associations between maternal educational level and offspring low birth weight and maternal educational attainment and SGA births, as calculated by the percentage variation of the odds ratio due to smoking during pregnancy. In a study conducted in France, Ballon and colleagues 13 also found that smoking during pregnancy was a significant mediator of the effects of low educational attainment in low birthweight and small offspring. Using structural equation models, Da Silva and colleagues found that maternal socioeconomic position indirectly influenced child birthweight through maternal and paternal smoking during pregnancy in Brazil. [START_REF] Silva Aam Da | Socioeconomic status, birth weight, maternal smoking during pregnancy and adiposity in early adult life: an analysis using structural equation modeling[END_REF] Therefore our data are largely consistent with research conducted in other settings, which did not apply the same rigorous method to test causal inference as we did.

Potential underlying mechanisms of smoking during pregnancy in the effects of maternal educational attainment on offspring birthweight for GA

This study contributes to the growing body of literature supporting the hypothesis of embodiment of social inequalities. Women with a more disadvantaged educational attainment are more likely to smoke tobacco during pregnancy, [START_REF] Hiscock | Socioeconomic status and smoking: a review[END_REF] and less likely to quit smoking. [START_REF] Schneider | Smoking cessation during pregnancy: a systematic literature review[END_REF] In a systematic review and a small meta-analysis, financial incitation on disadvantaged women has shown a potential positive effect on smoking cessation. [START_REF] Bryant | A systematic review and meta-analysis of the effectiveness of behavioural smoking cessation interventions in selected disadvantaged groups[END_REF] Our results also suggest an important effect of smoking during pregnancy on reducing offspring birthweight for GA, even after adjusting for a large number of potential confounders, including maternal educational attainment. This is in accordance with a substantial body of scientific literature reporting an association between maternal tobacco smoking and offspring birthweight for GA. [START_REF] Pereira Pp Da | Maternal active smoking during pregnancy and low birth weight in the Americas: a systematic review and meta-analysis[END_REF] Nicotine crosses the placenta and foetal nicotine concentrations could be increased by 15% of maternal (blood) concentrations, and could affect foetal growth and birthweight. [START_REF] Knopik | The epigenetics of maternal cigarette smoking during pregnancy and effects on child development[END_REF] The effect of maternal tobacco smoking during pregnancy on birthweight for gestational age could also reflect gene-environment interactions between tobacco use and genes that characterize the nicotine receptor and are associated with nicotine dependence. Women with low educational attainment are also more likely to face stress events during pregnancy such as being single mothers or having shift work, which increase their probability of not quitting smoking during pregnancy and of having smaller babies. [START_REF] Wolff | Prevalence and predictors of maternal smoking prior to and during pregnancy in a regional Danish population: a cross-sectional study[END_REF]

Strengths of the study

In terms of strengths, it is worth pointing out that our analyses are based on a large birth cohort study where children's birthweights were ascertained at the maternity ward and documented in medical records and controlled for a large number of potential confounding variables. Moreover, birthweights were assessed using z-scores which take into account GA and sex of the offspring. In addition, the assessment of the associations between maternal educational attainment, tobacco smoking during pregnancy and offspring birthweight for GA remained stable after adjustment for potential confounders, despite the important number of missing values. Finally, the use of mediation models, particularly wellsuited for the study of mechanisms underlying socioeconomic inequalities in health, is novel.

Limitations of the data

Our study has a number of limitations which should be considered. First, maternal educational attainment has been used as a proxy of socioeconomic position (SEP) even though SEP is a complex construct including other dimensions then education. However, maternal educational attainment have been shown as a good proxy to study the effect of SEP on children's health [START_REF] Kramer | Determinants of low birth weight: methodological assessment and metaanalysis[END_REF] as most financial resources result from salaries, thus from work, which access is often conditioned by educational attainment. [START_REF] Kramer | Determinants of low birth weight: methodological assessment and metaanalysis[END_REF] Then, ELFE is a cohort study based on voluntary participation. As a result the proportion of mothers with a low education level is lower than in the general population of France. Maternal smoking during pregnancy was self-reported at birth, which could lead to recall and desirability bias. However, the prevalence of maternal smoking in pregnancy in ELFE (approximately 20%) is consistent with national figures. [START_REF] Andler | Baromètre santé 2017. Consommations d'alcool et de tabac durant la grossesse[END_REF] Our measure of smoking during pregnancy did not take into account the frequency or the quantity of tobacco smoker. Sensitivity analyses on self-reported tobacco smoking in the third trimester of pregnancy (17% of the sample) lead to similar results as the ones regarding the whole pregnancy (data not shown) but women who smoked only in early pregnancy might have declared they did not smoke during pregnancy. We may have underestimated social inequalities in birthweight for GA but our results are generalizable. Second, our analyses were based on complete cases, resulting in the exclusion of 5,500 participants. Mothers who were excluded due to missing values were younger, had lower educational attainment and were more likely to be non-French citizens. The direction of bias resulting from this exclusion is not obvious. Of note, only children born at 33 weeks of gestation or above were recruited, thereby limiting the results to babies not born severely premature. Severe prematurity [START_REF] Knopik | The epigenetics of maternal cigarette smoking during pregnancy and effects on child development[END_REF] have other determinants and it is not clear if the contribution of tobacco smoking to social inequalities in this area would be similar as we observed. We thus acknowledge that the contribution of tobacco smoking to socioeconomic inequalities in infant birthweight should be examined in studies that include children born at younger gestational ages. Third, in France, levels of maternal tobacco smoking in pregnancy are relatively high (20% prevalence), but comparable to several other settings including Denmark and the Netherlands and some US States (e.g. West Virginia, Kentucky, Montana, Vermont, Missouri), which also calls into question the generalizability of our findings [START_REF] Momen | Exposure to maternal smoking during pregnancy and risk of childhood cancer: a study using the Danish national registers[END_REF][START_REF] Smedberg | Characteristics of women who continue smoking during pregnancy: a cross-sectional study of pregnant women and new mothers in 15 European countries[END_REF] .

Fourth, a simple mediation analysis was conducted, which might conceal the contribution of other potential mediators. Nevertheless, it is unlikely that these other mediators (e.g. maternal nutrition), would confound the role of tobacco smoking. Tobacco smoking during pregnancy was self-reported by the mother and could be underestimated, even though our estimations of tobacco smoking during pregnancy are similar to those obtained in other French studies. [START_REF] Andler | Baromètre santé 2017. Consommations d'alcool et de tabac durant la grossesse[END_REF][START_REF] Melchior | Maternal tobacco smoking in pregnancy and children's socio-emotional development at age 5: The EDEN mother-child birth cohort study[END_REF] Finally, sensitivity analyses pointed out some limitation to any causal interpretation of the observed interpretation between maternal educational attainment, maternal tobacco smoking and offspring birthweight for GA. Especially, we found a E-value of 1.3 [95%CI 1.2, +inf[ for the association between maternal educational attainment and offspring birthweight for GA, meaning any unmeasured factor being associated with a strength of a RR greater or equal to 1.4 could explain away the observed association between these two variables, after adjustment for measured potential confounders. Such factor could be any factor related to maternal diet during pregnancy for example.

Our study shows that the effects of maternal educational attainment on offspring birthweight for GA are in large part (39%) mediated by tobacco smoking during pregnancy. These data contribute to existing evidence that reducing maternal tobacco smoking during pregnancy could improve offspring as well as maternal perinatal outcomes, including birthweight for GA, and contribute to reducing socioeconomic inequalities in children's perinatal health. Interventions encouraging female smokers -particularly those who belong to socioeconomically disadvantaged groups -to quit smoking prior to or during pregnancy should be a public health priority across industrialized countries. However, more specific psychosocial interventions targeting women who are more at risk of smoking during pregnancy and having children with low birthweight for GA should be developed. These interventions should take into consideration women's socioeconomic and psychosocial characteristics, such as their ability to access social workers for improving their living conditions and to get support from their own network and health professionals during their pregnancy and after child birth. Crude OR 1 was calculated on total sample size while crude OR 2 on multivariable complete case sample.

Adjustment for age at birth, nationality, parity, residential area, relationship status, body mass index (BMI) before pregnancy, attendance of at least one class to prepare for childbirth, number of prenatal visits, severe psychological difficulties during, alcohol consumption during pregnancy, pregnancy complications, gestational weight gain, physical activity during pregnancy, maternity type and child sex.

  The ELFE study received the approval of France's bodies regulating ethical research conduct (Comité Consultatif sur le Traitement des Informations pour la Recherche en Santé: CCTIRS; Commission National Informatique et Libertés: CNIL).

Figure 1 -

 1 Figure 1 -Diagram acyclic graph (DAG), maternal educational attainment, maternal tobacco smoking during pregnancy, offspring's birthweight for gestational age and maternal prepregnancy body mass index

Figure 2 -

 2 Figure 2 -Crude and adjusted risk ratios and odds ratios for the association between 1) offspring birthweight for gestational age and maternal educational attainment 2) offspring birthweight for gestational age and smoking during pregnancy 3) smoking during pregnancy and maternal educational attainment (ELFE study, France, 2011)

Figure 3 -

 3 Figure 3 -Mediation analysis between maternal educational attainment (exposure), maternal smoking during pregnancy (potential mediator) and offspring birthweight for gestational weight (outcome), A) for underweight women (BMI < 18.5kg/m 2 , n=1,117) B) for women with a normal BMI (18.5 ≤ BMI < 25.0kg/m 2 , n= 9,520) C) for overweight women (25.0 ≤ BMI < 30.0kg/m 2 , n= 2,495) D) for obese women (BMI ≥ 30.0kg/m 2 , n=1,406), ELFE French Study, 2011

Figure 1S -

 1S Figure 1S -Mediation analysis between maternal education (exposure), smoking during pregnancy (potential mediator) and offspring birthweight for gestational age (outcome) (ELFE Study, France, n=14,538, 2011) PM = proportion mediated

  E-values were estimated at 1.32 [95%CI 1.16, +inf[ for adjusted maternal educational attainment and offspring birthweight for gestational age association; 2.08 [95% CI 1.96, +inf[ for maternal tobacco smoking and offspring birthweight for GA and 2.59 [95%CI 2.44, + inf[ for maternal educational attainment and maternal tobacco smoking associations.

Table 1 -

 1 Adjusted risk ratios (RR) and odds ratios (OR) for the association between 1) offspring birthweight for gestational age and maternal educational attainment 2) offspring birthweight for gestational age and smoking during pregnancy 3) smoking during pregnancy

	and maternal educational attainment stratified by body mass Index (BMI) in four categories
	(<18.5; 18.5-<25;25-<30; ≥ 30kg/m 2 ), ELFE Study, France, 2011
	Exposure/Outcome	Offspring birth weight	Maternal smoking
		for gestational age	during pregnancy
		Adjusted RR [95% CI]	Adjusted OR [95% CI]
	BMI < 18.5kg/m2, n=1,117
	Maternal educational level	0.82 [0.72, 0.93]	3.17 [2.30, 4.38]
	Maternal smoking during pregnancy	0.78 [0.69, 0.88]
	18.5 ≤ BMI < 25.0kg/m2, n=9,520
	Maternal educational level	0.95 [0.91, 0.99]	2.66 [2.56, 2.99]
	Maternal smoking during pregnancy	0.72 [0.69, 0.76]
	25.0 ≤ BMI < 30.0kg/m2, n=2,495
	Maternal educational level	0.96 [0.88, 1.04]	2.09 [1.66, 2.65]
	Maternal smoking during pregnancy	0.70 [0.63, 0.77]
	BMI ≥ 30.0kg/m2, n=1,406
	Maternal educational level	0.95 [0.84, 1.07]	2.27 [1.66, 3.10]
	Maternal smoking during pregnancy	0.77 [0.66, 0.87]
	Adjustment for age at birth, nationality, parity, residential area, relationship status,
	attendance of at least one class to prepare for childbirth, number of prenatal visits, severe
	psychological difficulties during, alcohol consumption during pregnancy, pregnancy
	complications, gestational weight gain, physical activity during pregnancy, maternity type
	and child sex.

Funding

The Elfe survey is a joint project between the French Institute for Demographic Studies (INED) and the National Institute of Health and Medical Research (INSERM), in partnership with the French blood transfusion service (Etablissement français du sang, EFS), Santé Publique France, the National Institute for Statistics and Economic Studies (INSEE), the Direction générale de la santé (DGS, part of the Ministry of Health and Social Affairs), the Direction générale de la prévention des risques (DGPR, Ministry for the Environment), the Direction de la recherche, des études, de l'évaluation et des statistiques (DREES, Ministry of Health and Social Affairs), the Département des études, de la prospective et des statistiques (DEPS, Ministry of Culture), and the Caisse nationale des allocations familiales (CNAF), with the support of the Ministry of Higher Education and Research and the Institut national de la jeunesse et de l'éducation populaire (INJEP). Via the RECONAI platform, it receives a government grant managed by the National Research Agency under the "Investissements d'avenir" programme (ANR-11-EQPX-0038).

Additionally, this analysis was funded by the SOCIALRISK_MH project funded through the ANR 'Social determinants of health' program (2012), where Maria Melchior is also the Principal Investigator.

Conflicts of interest

The authors declare that they have no competing interests

Key points


Vincent Rouger

Guillaume Bordet

Carole Couillault

Serge Monneret

Sébastien Mailfert

Jonathan J Ewbank

Nathalie Pujol
email: pujol@ciml.univ-mrs.fr

Didier Marguet
email: marguet@ciml.univ-mrs.fr

Independent synchronized control and visualization of interactions between living cells and organisms

Keywords: Biophotonic control of C. elegans infection Holographic optical tweezers, spinning disk confocal microscopy, cell-cell interaction, infection, C. elegans 2

come   L'archive ouverte pluridisciplinaire

Cell-cell contacts are key elements of regulation in diverse biological systems, such as neuronal networks (1), immune synapses (2,[START_REF] Monks | Threedimensional segregation of supramolecular activation clusters in T cells[END_REF] or between host and pathogen [START_REF] Stuart | Phagocytosis and comparative innate immunity: learning on the fly[END_REF]. Delineating the molecular mechanisms involved in these processes is still challenging since there is a need to record the sequence of events occurring at the early steps with appropriate spatio-temporal resolution. In this context, the use of trapping techniques to manipulate and control the initial contact events between cells hold much promise. Among these techniques, optical tweezers can be used to manipulate biological systems without damage [START_REF] Wang | Visualizing the mechanical activation of Src[END_REF][START_REF] Khalil | Single M13 bacteriophage tethering and stretching[END_REF][START_REF] Li | Membrane tether formation from outer hair cells with optical tweezers[END_REF][START_REF] Kim | The alphabeta T cell receptor is an anisotropic mechanosensor[END_REF][START_REF] Mohanty | Dynamics of Interaction of RBC with optical tweezers[END_REF]. Optical tweezers were first described in the seminal paper of Ashkin et al [START_REF] Ashkin | Observation of a singlebeam gradient force optical trap for dielectric particles[END_REF] in which the authors provided the first demonstration that a single laser beam focused through a lens can trap mesoscopic objects [START_REF] Ashkin | Observation of a singlebeam gradient force optical trap for dielectric particles[END_REF][START_REF] Ashkin | Optical trapping and manipulation of neutral particles using lasers[END_REF]. The main advantage of using optical tweezers lies in limited mechanical stress and allows precise and fast object manipulation.

Trapping techniques in general suffer, however, from a lack of resolution, especially if one considers the need for simultaneous 3D imaging during the trapping period. In part this is because most optical trapping systems are built around epifluorescent or total internal reflection fluorescence microscopy systems for data acquisition [START_REF] Grashoff | Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics[END_REF]. To overcome this issue, optical trapping system have been developed that combine tweezers with a confocal or 2 photon imaging module [START_REF] Oddos | High-speed high-resolution imaging of intercellular immune synapses using optical tweezers[END_REF][START_REF] Heinrich | Dynamic sorting of lipids and proteins in membrane tubes with a moving phase boundary[END_REF][START_REF] Goksor | Optical manipulation in combination with multiphoton microscopy for single-cell studies[END_REF][START_REF] Hoffmann | Control and manipulation of pathogens with an optical trap for live cell imaging of intercellular interactions[END_REF](17). Such systems significantly improve the spatial resolution but are still interdependent: the focal plan of imaging is directly linked to that of the traps. More recently, independent trapping and imaging modules have been combined but with two objectives in an upright-inverted configuration [START_REF] Yevnin | Independent and simultaneous three-dimensional optical trapping and imaging[END_REF].

In the present study, and because we needed to record from biological samples with 3-dimensional resolution, we have designed a versatile setup optimized for general biological applications on cellcell interactions. It combines two independent modules, one for trapping and the other for imaging. The trapping module has been described previously [START_REF] Belloni | Quadrant kinoform: an approach to multiplane dynamic three-dimensional holographic trapping[END_REF]. It is based on holographic optical tweezers (HOT) using a spatial light modulator (SLM), allowing different possibilities to move multiple trapped objects independently in the three directions [START_REF] Belloni | Quadrant kinoform: an approach to multiplane dynamic three-dimensional holographic trapping[END_REF] as well as rapid and long distance displacement of traps using speeds of up to 500 ȝm/s. The second module comprises a versatile fast confocal imaging system based on a Nipkow spinning disk system. Here, we provide a complete decoupling of the two modules by correcting the trap focalization during the scanning of 3D objects. It is noteworthy that the two modules use the same objective, thereby providing a simpler access to biological samples in comparison to a two-objective configuration [START_REF] Yevnin | Independent and simultaneous three-dimensional optical trapping and imaging[END_REF].

As a proof of principle, we use infection of Caenorhabditis elegans by the fungus Drechmeria coniospora to illustrate the potential of this setup. For the first time, we analyze precisely the initiation of adhesion between the fungal spores and the worms by testing the first contact of single spores to different region of the worm and on different stages of development. Finally, by using multicolor acquisition and multiple traps at the same time, we demonstrate how this setup is suitable for following the early steps of the infection and could be used to investigate the molecular signal transduction cascade induced by this infection.

Materials & methods

Biological material

All C. elegans strains were maintained on nematode growth media and fed with E. coli strain OP50, as described [START_REF] Stiernagle | Maintenance of C. elegans. WormBook : the online review of C. elegans biology[END_REF]. In addition to the wild-type strain N2 that was obtained from the Caenorhabditis Genetics Center (CGC), the following mutants were used: bus-2(e2687) IV, bus-4(e2693) IV, bus-12(e2977) IV, bus-17(e2800)X [START_REF] Gravato-Nobre | Glycosylation genes expressed in seam cells determine complex surface properties and bacterial adhesion to the cuticle of Caenorhabditis elegans[END_REF], the kind gift of Jonathan Hodgkin, as well as the transgenic strains IG1256 (CHC-1::GFP; MYO-2::RFP) [START_REF] Dierking | Unusual regulation of a STAT protein by an SLC6 family transporter in C. elegans epidermal innate immunity[END_REF]. Methods for infecting C. elegans with D. coniospora were as previously described [START_REF] Pujol | Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides[END_REF].

The 3A9 T cell hybridoma is growth as described in the supporting Material (see Method M1).

Sample preparation for imaging

Worms were anesthetized with levamisole (Fisher scientific, Illkirch, France) in 50 mM NaCl at a concentration that depended on the worm's developmental stage (between 0.01 and 0.005 %). Following removal from levamisole, all worms recovered a normal mobility after 10 min to 6 h. For some experiments, the worm cuticle was labeled by incubation for 30 min at 20°C with a 10 ȝg/ml solution of DiD (Life Technologies, Saint-Aubin, France) in 50 mM NaCl.

When needed, D. coniospora spores (10 7 /mL) were labeled with DiI (Life Technologies, Saint-Aubin, France) at a final concentration of 20 ȝg/ml in 50 mM NaCl for 15 min at 20°C, then washed in the same buffer.

All trapping experiments are done in the anesthetic solution. For the infection experiments, a sample holder was designed (Fig. S1 in the supporting Material) to avoid uncontrolled worm infection by spores diffusing within the medium. Two adjacent wells (one for the worms, the other for the spores) are connected by a small channel (1 × 0.5 × 0.1 mm) allowing spores to be dragged from one side to the other with the optical tweezers. Due to a strong adherence of the spores to the cover glass, we add spores repeatedly to the reservoir to keep some of them in suspension. Each experimental condition was evaluated for 10 different spores. The experiments were performed at 21°C on five worms, with no more than 2 spores per worm.

Dynamic HOT combined with independent fast 3D-confocal imaging setup

The fast confocal imaging module was combined with HOT around a motorized double-stage inverted microscope (Nikon France SAS, Champigny-sur-Marne, France) (Fig. 1 A). The lower level of the stage microscope (M1) is used for confocal imaging, direct visualization and brightfield imaging, the upper one (D1) for FRAP and fluorescence wide-field illumination and we added a third one (D2) for the HOT module.

The microscope is equipped with a halogen light, a DIC contrast polarizer, a Wollaston prism and a electromechanical shutter (Uniblitz, Polytec, Châtillon, France) for fast contrast imaging during multicolor acquisition. Two objectives (Nikon, CFI 4 ×, 0.1 NA) and (Nikon, CFI Plan 10 ×, 0.25 NA) are used for HOT alignment and sample location, the two others (Nikon, CFI Plan Fluor 40 × oil, 1.3 NA) and (Nikon, CFI Plan Fluor 100 × oil, 1.3 NA) for recording and trapping. A xy motorized stage (ASI, Optophase, Lyon, France) is combined with a piezo z-top plate for 3D acquisition (ASI). Four different light pathways (100 % binocular, 100 % left, 100 % right and a modified base port for 20 % binocular and 80 % right) are set on the microscope stage. The microscope environment is regulated by a temperature controller box (Digital Pixel, Optophase, Lyon, France). The whole system is built on an optical breadboard (Melles Griot Industrie, Voisinsle-Bretonneux, France).

Trapping module -The trapping setup comprises two main optical components, a 1050 nm laser (IPG, Burbach, Germany) to create the trap and a programmable SLM (Hamamatsu Photonics France, Massy, France) to control in real time multiple traps in 3D. The direction of the IR light polarization is tuned by a half-wave plate (Ȝ/2 wave plate) (Thorlabs SAS France, Maisons-Laffite, France) to fit the SLM liquid crystal polar orientation. Two lenses L1 (f = 30 mm) and L2 (f = 120 mm) (Thorlabs) expand the laser beam diameter to overfill the SLM. The reflected laser beam is guided by an adjustable second telescope made of two lenses L3 (f = 150 mm) and L4 (f = 125 mm) (Thorlabs) to fill the back aperture of the trapping objectives and to minimize the loss of power. A perfect reproduction of the SLM hologram at the back focal plane of the objective required a 4f-configuration of the L3L4 telescope. The linear motor LM (PhyskInstrumente, PI France SAS, Montrouge, France) controls the L3 position to dissociate the focal plane of imaging from the trap position. The light is reflected on the D2 infrared dichroic mirror (Rocky Mountain Instrument Co., Lafayette, CO USA). The infrared filter F1 (Thorlabs) is duplicated to block efficiently IR light on the collection path. Images from the CCD camera (JAI, Stemmer imaging SAS, Suresnes, France) on the right side port of the microscope allow to monitor traps on the sample by a custom-made software [START_REF] Belloni | Interactive holographic optical tweezers for the dissection of membrane signaling processes[END_REF].

Imaging module -This setup adapted by Visitron (Visitron Systems GmbH, Puchheim, Germany) is composed of three modules. The first one encompasses six laser sources: 405 nm, 440 nm, 473 nm, 488 nm, 561 nm and 640 nm lines. All lasers except the 473 nm laser line are combined (AMS Technologies SARL, Les Ulis, France) in the fiber of the spinning disk module for confocal imaging. The 473 nm laser line is collimated with part of the 405 nm laser in an independent fiber (AMS Technologies) for FRAP experiments. The second module i.e., the confocal spinning disk head (Yokogawa, Visitron Systems GmbH) is on the left side of the microscope. Two 512 × 512 pixels EM-CCD cameras (Hamamatsu) are set for simultaneous two-color detection. The third module (Visitron) dedicated to FRAP measurement has been implemented on the same port as the mercury lamp (Nikon) by using a double lamphouse adapter M2. A real time FRAP scanner module provides the possibility to perform classical spot and ROI FRAP or "FRAP on the fly" measurements during trapping experiments.

Data acquisition

For brightfield or fluorescence image recording, the laser power, the integration time and gain of the camera were adjusted to optimize the contrast. 3 ȝm latex beads (Polyscience Europe GmbH, Eppelheim, Germany) were used for LM calibration. For D. coniospora trapping, a laser power set at 60 mW at the focalization point allowed us to drag the spores at a maximum speed of 0.5 mm/s.

Results and Discussion

Decoupling HOT from fast 3D confocal imaging

Our aim was to combine HOT with fast 3D confocal imaging in order to observe biological samples before, during and after the initial contact with trapped objects (Fig. 1 A). Our first experimental setup kept the image acquisition and the trap within the same focal plan (Fig. 1 B left panel). The motion of the z-top plate supporting the samples permits the acquisition of 3D images but the trapped samples moved at the same time. As a consequence, it was not possible to dissociate the confocal plane from the trapping one, nor to record a 3D stack of images of the trapped object (Fig. 1 B left panel and 2 A). Here, we implement a system to keep traps independent from the imaging module (Fig. 1 B right panel and 2 B). The details of the setup are described in the Materials and Methods section. This required the position of traps within the sample to be held static and therefore, to change the focalization distance of the IR laser. Timelapse imaging forces to adjust the distance of the traps sufficiently rapidly to match the maximum frame rate of the imaging system, for instance at 30 ms in full-frame in the present case. Moreover, to reach appropriate spatiotemporal resolution for live imaging, one wants to record 10 focal planes in less than 1 s for 3D-image reconstruction of 10 ȝm thick objects.

One possible solution lies in refreshing the hologram of the SLM and synchronizing it with the detection frame rate. The calculation of hologram patterns is limited, however, to 2 to 5 Hz when performed in real time, a frequency incompatible with the required image acquisition rate. Alternatively, it is possible to move one of the HOT lenses, as long as it can be operating in synchrony with the imaging module. As a first estimation, a one micrometer displacement of the sample from the focal plane would imply a lens movement of around 2 mm. Therefore, repeated lens displacement with a linear motor (LM) at a frequency of 30 Hz allows the synchronization of the trapping and imaging modules. In principle, the LM is run as a slave of the imaging system by external voltage control. Consequently, this latter option adequately fits the main constraints required to achieve the appropriate temporal resolution for live imaging.

Servo control of the lens displacement

Overfilling the SLM active area implied blocking L1 and L2 lenses at a specific distance. Moreover, our setup was not compatible with the addition of a motor to move L4 due to space limitation on the optical bench. Consequently, we had the possibility of only adding a motor to the L3 lens (Fig. 1 A). This LM has the capability to operate a 2 mm displacement in less than 30 ms (Fig. S2).

The LM has to compensate the sample holder displacement in real time by correcting the position of the L3 lens to keep the traps at their initial positions during the acquisition of a 3D-stack of images. The displacement has to be calculated for a continuum of positions. We therefore determined the equation linking the position of the sample holder to the one of L3. We first measured experimentally the LM displacement for different sample holder positions (from 0 to 30 ȝm for the 40 × objective). The LM position is determined by imaging latex beads in brightfield (Fig. 3 A); their shape provided an accurate evaluation of their z-position as described below. All of the experimental data were fitted with Eq. 1 except the 100 ×/100 mm data fitted with Eq. 2. Fitting parameters are configured in the synchronization module of the Visiview software (Fig. S3).

The overlap of the imaging system and traps' focal planes defined the reference positions of the two actuators, the z-top plate controlling the sample position and the LM controlling the L3, respectively. This reference initial position was achieved by tuning the L3 position in order to optimally trap a reference bead that had settled on the cover glass. Next, we moved the sample holder by steps along the optical axis and controlled L3 to maintain the trapped bead on the cover glass, by matching on the CCD image the shape of the trapped bead with the ones of non-trapped settled beads. By iteration, we drew an experimental curve of the L3 displacement as a function of the sample holder position on the optical axis (Fig. 3 B). The linear equation Eq. 1 efficiently fits the experimental data from which a and b parameters were estimated for a given set of lenses and objective. Once the values are filled up within the Visiview software (Fig. S3), the L3 position is automatically calculated to compensate the sample displacement in real time.

݂ሺݔሻ ൌ ܽ ܾ ൈ ݔ

(1)

General consideration on the optical setup

Some biological applications might require a range of xy magnification, a z-stack accessible range for optimized trapping forces, and depth field exploration. For instance, the maximal 50 mm LM course limits the thickness of the z-stack accessible range to 15 ȝm and 40 ȝm with the 100 × and 40 × objectives, respectively (Fig. 3 B). This means that higher magnification requires more L3 translation to compensate a given z-top plate displacement, but reduces the image sampling. Moreover, the longer the focal lenses are, the longer the L3 translation is required to compensate the same z-top plate displacement (Fig. 3 B).

In fact, different combination of optical lenses and objective will critically influence the servo control of L3 displacement and in some case, the fitting model linking the displacements between actuators. For example, we observed with the 100 × objective a nonlinear curve when using a 100 mm focal length for L4 (Fig. 3 B). In that case, an exponential equation, Eq. 2, fits efficiently the data:

݂ሺݔሻ ൌ ܽ െ ܾ ൈ ݁ ି ௫ (2)
A L3/L4 set of 150/100 mm focal lengths fits more perfectly the back aperture of the objective and implies smaller LM displacement. We have been constrained, however, by a space limitation on the optical bench to use a L3/L4 set of 150/125 mm focal lengths.

Moreover, the movement of L3 slightly decollimates the IR laser beam at the back aperture of the objective. We have evaluated this effect on the trapping force F by measuring it with the drag force method on a spherical object (Fig. S4), and quantifying it by the following equation Eq. 3:

ܨ ൌ ߨ ߟ ݎ ݒ (3)
in which Ș is the viscosity constant of the medium, r the radius of the bead, v the maximum velocity [START_REF] Belloni | Interactive holographic optical tweezers for the dissection of membrane signaling processes[END_REF][START_REF] Capitanio | Calibration of optical tweezers with differential interference contrast signals[END_REF]. The variations of F result from two effects: (1) the angle of incidence of the photons coming out varies with the distance of trapping from the objective. Consequently, the closer the trap is, the higher the trapping force is; (2) the diameter of the beam filling the back aperture of the objective varies with the movement of L3 lens. It increases or decreases when the trapping plane is above or below the imaging focal plane, respectively. This has a direct effect on the quantity of photons reaching the trapped bead, and consequently causes a change in the trapping force. But from our own observations, this did not affect the ability to trap objects such as bacteria, beads, spores or eukaryotic cells along the required z-axis range.

In the following biological experiments, we have performed the observations with the optical setup described in the Materials & Methods section and the 40 × objective.

Trapping movement possibilities with spores for different imaging modes

Depending on the biological models and questions, users can have different needs. To provide the most versatile solution, the system was developed to increase the number of possible manipulations for the trapped sample. First, using the motors of the sample holder with a fixed trapped sample in the imaging field and with the displacement of the sample on the cover glass (Fig. 4 A) allows users to move the trapped sample for long distances, in the range of cm for motors and in the micrometer range along the optical axes. These fast displacements give the possibility to bring two biological objects together from different sides, preventing the risk of unwanted contacts. But using this displacement, one is not able to move the different trapped objects independently in 3D. Second, we can use the SLM to move trapped objects without changing the field of view on the cover glass (Fig. 4 B). This fine displacement gives a high precision in time and space in the manipulation, of less than one micrometer (~250 nm) in all directions. This also allows one to move independently more than 10 different trapped objects in the optical plane, or in 3 different focal planes. These two different ways to move objects can be used at the same time and during imaging, independently of the imaging mode (2D or 3D). While we show here the versatility of the system using fungal spores, different biological objects can be trapped, like functionalized beads or different cells types, using either objects in suspension or ones that are adherent to a surface.

Characterization of spore adhesion to C. elegans

D. coniospora is a nematophagous fungus that infects C. elegans and other species of nematodes. D. coniospora produces adhesive conidia or spores that attach to the worm's cuticle. These germinate to produce invasive hyphae that penetrate the cuticle and grow throughout the epidermis [START_REF] Dijksterhuis | Ultrastructural study of adhesion and initial stages of infection of nematodes by conidia of Drechmeria coniospora[END_REF][START_REF] Jansson | Life cycle of the endoparasitic nematophagous fungus Meria coniospora: a light and electron microscopic study[END_REF]. In C. elegans, infection with D. coniospora provokes in few hours an innate immune response in the epidermis involving the expression of a large number of defense genes [START_REF] Pujol | Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides[END_REF][START_REF] Couillault | TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM[END_REF][START_REF] Engelmann | A comprehensive analysis of gene expression changes provoked by bacterial and fungal infection in C. elegans[END_REF][START_REF] Engelmann | Innate immunity in C. elegans[END_REF]. If this host response has been well described, less is known about the very first step of the adhesion of the spore to the worm's cuticle, and the molecular nature of this interaction. It has been previously shown that the adhesion of spore is not homogeneous on the adult, and that they have a preferential adhesion to the tip of the head and to the vulva [START_REF] Jansson | Life cycle of the endoparasitic nematophagous fungus Meria coniospora: a light and electron microscopic study[END_REF][START_REF] Jansson | Adhesion of Conidia of Drechmeria coniospora to Caenorhabditis elegans Wild Type and Mutants[END_REF]. Adhesion tests on another species of nematode indicated no involvement of carbohydrates in the adhesion process and suggested that the adhesion is mediated by protein(s) in the adhesive part of the conidium binding to protein(s) excreted from the sensory organs of the nematodes [START_REF] Jansson | Adhesion to Nematodes of Conidia from the Nematophagous Fungus Drechmeria coniospora[END_REF]. Using C. elegans, it was shown that the adhesion was reduced after exposure of the worm to Pronase E indicating the requirement of proteinaceous material [START_REF] Jansson | Adhesion of Conidia of Drechmeria coniospora to Caenorhabditis elegans Wild Type and Mutants[END_REF]. All these studies were done using a concentrated solution of spore and observing a population of randomly infected worms after several hours of infection.

As a test of the versatility of the system described above, we decided to investigate precisely the adhesion of spores of the fungus D. coniospora to C. elegans, by carefully controlling the contact of single spores to different regions of the worm and at different stages of development. In order to do so, we first developed a simple 2-well chamber to keep spores separated from the worms until required (Fig. S1). We then used the HOT to move individual fungal spores into contact with the surface of individual worms. By using two simultaneous traps on a single spore, we were able to orient the spore's contact with the worm. As expected, we observed that the adhesive bud was the only part of the spore that adhered (data not shown). Using a single trap, spores were orientated within the optical axis. By adjusting the z position of the adhesive bud to bring it into the focal plane of the imaging system, we could show that the head, vulva and tail region of the adult hermaphrodite worm as well as the male fan allow an immediate adhesion (Table 1 and Fig. S5). This adhesion had such a strength that it could not be broken using the optical tweezers in the range of several hundreds of pN as estimated with beads (data not shown). If spores adhered efficiently to the head at all stages, they did so to the body only at the L4 and young adult stage.

The outermost layer of the nematode cuticle is called the surface coat, and is predominantly made up of glycoproteins. It has been shown that its composition in glycans varies with developmental stage and differs over the surface of the nematode [START_REF] Davies | Cuticle surface coat of plant-parasitic nematodes[END_REF]. The pathogenic bacterium Microbacterium nematophilum attachs to the cuticle via specific glycans [START_REF] Gravato-Nobre | Multiple genes affect sensitivity of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum[END_REF][START_REF] Yook | Mos1 mutagenesis reveals a diversity of mechanisms affecting response of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum[END_REF]. To test whether glycans could also be involved in the first contact adhesion of the fungal spore with the cuticle of the worms, we took advantage of known mutants in genes encoding different classes of galactosyltransferase (bus-2, bus-4 and bus-17) and a sugar transporter (bus-12) that were isolated in screens for mutants resistant to M. nematophilum [START_REF] Gravato-Nobre | Glycosylation genes expressed in seam cells determine complex surface properties and bacterial adhesion to the cuticle of Caenorhabditis elegans[END_REF][START_REF] Gravato-Nobre | Multiple genes affect sensitivity of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum[END_REF][START_REF] Yook | Mos1 mutagenesis reveals a diversity of mechanisms affecting response of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum[END_REF]. The attachment of M. nematophilum to these mutants is much reduced. Unexpectedly, detailed analyses of the adhesion of spores to the bus mutants using the HOT system revealed that the body region of bus-2 bus-12 and bus-17 adult worms is more adhesive than that of the wild type (Table 2). Probably as a consequence, the 3 mutants were more susceptible to D. conosporia infection (not shown). A detailed biochemical analysis of bus-2 mutant worms has shown that the level of core-1 glycans is decreased, whereas that of fucosyl Oglycans is increased. As a confirmation, in the same study it was shown that there was an increased staining of the bus-2 mutant cuticle with an Į linked L-fucose-specific lectin, (U. europaeus agglutinin I). Interestingly, this lectin also stained the vulva of wild type worms [START_REF] Palaima | The Caenorhabditis elegans bus-2 mutant reveals a new class of O-glycans affecting bacterial resistance[END_REF]. These results raise the possibility that fucosyl glycans are a target for spores binding.

TABLE 1 D. coniospora adhesion on C. elegans at different stages of development

The adhesion tests were performed on different area of hermaphrodites or males at different stages of development as delineated in Fig. S5. Results are expressed as the number of positive adhesion of a single spore on worms over the total number of trials. A test is positive (1) if adhesion occurred during the course of HOT contacts between spore and worm (usually, a single contact is sufficient) and ( 2) if the association is stable enough to not be dissociated by HOT. a only a precursor of the vulva at this stage, b uncontrolled motion of the worm tail prevent the experiment.

TABLE 2 D. coniospora adhesion on adults or eggs of different mutants of C. elegans

Results are expressed as in Table 1 We then tested the 3D imaging during the trapping period and the adhesion of the spore. As a proof of concept, we used a transgenic strain of C. elegans that contains two different fluorescent reporters, one labeling the apical epidermis in GFP, the other an internal structure called the pharynx in RFP. We also stained the worm cuticle and sensory neurons with DiD and the fungal spores with DiI. We were able to drag and image in 3D the adhesion of a fungal spore in multiple colors (3 in this case), allowing a spatial resolution of the different subcellular entities of the worm (Fig. 5). This opens the possibility in the future to decipher the activation of the signaling pathways known to be acting during the infection process and to follow the subcellular events that occur after the first contact between the pathogen and its host.

Conclusion

In this work, we developed a setup combining dynamic optical tweezers with a fast 3D imaging confocal head in which we have made the two modules optomechanically independent from each other. This involved compensating in real time the displacement along the optical axis during 3D imaging to keep the trapped object in its initial position. This was done by an LM moving a lens in the light path of the trapping laser beam. We synchronized this motor with the z-scanning and the cameras. It provides access to all of the confocal imaging functionalities such as 3D multicolor imaging or FRAP measurements, while allowing multiple and independent manipulation of a large diversity of biological samples (Movie S6 and S7).

In comparison with other HOT/confocal imaging setups (13,17,18) (Table 3), our system extends the possibility to use HOT for the analysis of cell-cell interactions on very different biological systems. This is firstly due to the possibility to extend imaging from 2D (13,17) to 3D (( 18) and this work) while trapping objects. Moreover, as compared to (18), the present system uses a single objective for trapping and imaging, affording simple and efficient acquisition for routine experimentation. Although the trapping force is not rigorously constant during z-scanning due to slight decollimation of the IR laser beam, the traps conserve sufficient forces to drag multiple objects with a large range of speed.

We previously used our HOT module with classical 2D imaging for many different applications such as the study of protein recruitment, cell-cell adhesion (unpublished data T. Trombik, V. Rouger, S. Piatek, S. Mailfert, S. Ugolini, E. Vivier, D. Marguet), or nanotube formation between cells [START_REF] Rainy | H-Ras transfers from B to T cells via tunneling nanotubes[END_REF]. Now, we are able to perform 3D imaging for the same type of applications. Here, we demonstrate the interest of this method by analyzing the infection process of C. elegans by D. coniospora. The experiments reported here by using fluorescent transgenic strains of C. elegans highlight the possibility of using our setup to follow in real time the molecular cascades that accompany the early steps of infection. In future work, we will aim to use the possibilities of 3D imaging and multiple trapping to understand how C. elegans worms are able to integrate simultaneous or sequential infections by D. coniospora. In order to overcome undesired effects due to worm anesthesia, we aim to add a microfluidic system to mechanically constrain the worm during the infection process [START_REF] Gilleland | Microfluidic immobilization of physiologically active Caenorhabditis elegans[END_REF]. A latex bead of 3 μm in diameter is initially trapped in the imaging plane of the imaging, then moved by the linear motor above or below this plane from a given distance as reported on the ordinate axis. Next, the trapping force F is experimentally quantified by dragging the bead at increased speeds until its release from the trap. All measurements were performed in 50% glycerol medium at 30°C. The IR laser power at the reference position was set at 60 mW at the focalization point.

FIGURE 1

 1 FIGURE 1 Overview and schematic of the experimental setup. (A) Instrument layout showing the illumination path for brightfield (grey), fluorescence excitation and emission (dark blue, green & red) and FRAP and arc lamp (cyan) and the trapping infrared beam (orange). Mirrors (M1 and M2), dichroic mirrors (D1 & D2), Ȝ/2 wave plate (Ȝ/2), lenses (L1-L4), spatial light modulator (SLM), linear motor (LM), IR filter (F1) and CCD camera (CCD). The grey dotted line features the software synchronization between the trapping and imaging modules. Two EM-CDD cameras record filtered fluorescence signals coming from the spinning disk confocal module. (B) Representation of L3 motion effect on the trap focalization during 3D sample imaging.On the left side, the focal plane of the trapped object is not dissociated from the one of 3D imaging. On the right side, the displacement of L3 by LM is synchronized compensating the z displacement of the imaging module and keeping the trap at its reference position. At other positions than the initial one, the IR laser becomes decollimated, this modifies the trapping force (Fig.S4).

FIGURE 2

 2 FIGURE 2 Effect of uncoupling trap from imaging on the 3D image reconstruction of a trapped object. In (A) and (B), on the left panels, a single latex bead (arrow) from the ones settled down on the cover glass is trapped, moved by the z-top plate and imaged in brightfield; on the right panels, a CD45 membranestained T cell hybridoma (CD4 3A9 T cell) was trapped and observed by confocal imaging (stack of 20 images at 1 ȝm intervals). Scale bars, 15 ȝm. (A) The sample was moved by the z-top plate without synchronization (Fig. 1 B left panel). The trapped bead stays in the focal plane while beads on the cover glass are defocused. As a consequence, a trapped object is constantly viewed through the identical focal plane all along the stack as illustrated on the right panel with a 3D-image of a cell. (B) Same experiments but with the trapped object maintained in its reference position by synchronization (Fig. 1 B right panel). The trapped bead appears defocused as the ones on the cover glass. On the right, 3D image reconstruction of a trapped cell becomes possible from the stack of images recorded at different altitudes. See also Movie S1 and S2, on beads and cells, respectively.

FIGURE 3

 3 FIGURE 3 Calibration of the lens displacement compensating the z-top plate motion. (A) Brightfield images of a latex bead (3 ȝm in diameter) at different altitudes (scale bar, 5 μm). Out of focus position changes the image of the bead with a white or black centroid above and below the reference plane, respectively. These well-defined changes in the bead shape are used to control L3 position thank to additional beads settled on the cover glass. (B) Calibration data reported for different combinations of objectives (40 × or 100 × objective) and L3 lenses (100 or 125 mm focal length): 40 ×/100 mm (solid circle), 40 ×/125 mm (solid square), 100 ×/100 mm (open circle) and 100 ×/125 mm (open square).All of the experimental data were fitted with Eq. 1 except the 100 ×/100 mm data fitted with Eq. 2. Fitting parameters are configured in the synchronization module of the Visiview software (Fig.S3).

FIGURE 4

 4 FIGURE 4 Multiple spatiotemporal trajectories to analyze spore-worm interactions. (A) Sedated worms can be moved by the xyz stage of the microscope to contact a stationary trapped spore(s). In the case of multiple traps, they are all linked to each other. (B) Alternatively, trapped spore(s) can be moved in all directions by HOT. Moreover, each trap is independent both in space and time. Directional movements are figured by a red line in xy plane and by a circle in z axis; the direction by an arrow; multiple arrows represent different arrival times. Movie S3-S5 illustrate the different modes of analysis of D. coniospora adhesion on C. elegans.

FIGURE 5

 5 FIGURE 5Early adhesion of a single spore on C. elegans by 3D multicolor confocal imaging. An adult nematode carrying GFP and RFP markers in the apical epidermis and pharynx, respectively is visualized after staining its cuticle and neuron with DiD. A fungal spore stained with DiI (arrow) is trapped to contact the head area of the worm by HOT. The system allows the sample visualization in 3D before and during the adhesion process. See Movie S6.

Figure S2 -

 S2 Figure S2 -Speed and acceleration characteristics of the linear motor supporting L3All of the data were recorded and visualized with the software controlling the linear motor (LM) (PI France SAS, Montrouge, France). (A) The acceleration closed-loop control was set at its default value (200 mm/s 2 ) to test motor speeds ranging from 50 to a maximum of 200 mm/s. Considering the constraints due to the weight of the L3 lens, the latter speed value fits our requirement. (B) The speed control was set at its maximum value (200 mm/s) for comparison of the default and maximum closed-loop acceleration values, 200 and 4000 mm/s 2 , respectively. By reaching 2 mm displacement in less than 30 ms, the LM characteristics fit our requirement to synchronize the trapping and imaging modules. To avoid inconsistent error message due to small overshot at the deceleration, the maximum position error has to be set at 5 μm.

Figure S3 -

 S3 Figure S3 -Synchronization module implemented in the Visiview softwareThe windows for focus configuration display different options in the mode of acquisition requested (A) and a list of controlled devices with adjustable position settings. The list includes the synchronized mode between both motors (Synchronizer -Focus), the LM (Pi -Focus) and the microscope control of the objective position (Nikon -Focus). (B). In "Synchronizer -Focus" mode, a second window is open to determine master and slave and their initial positions. This window allows to choose a linear (C) or exponential (D) model for synchronization and to introduce appropriate equation parameters.

Figure S4 -

 S4 Figure S4 -Variation of the trapping force during the IR laser decollimation

Figure S5 -

 S5 Figure S5 -Different regions of C. elegans visualized by light microscopyStitching reconstruction of a hermaphrodite N2 wild-type worm imaged by differential interference contrast (DIC) microscopy. The vignettes detail the head, vulva and tail of the worm. The black boxes delineate the area of adhesion tests. The body area is defined by excluding tail head or vulva with a security distance of around 100 μm. The upper right image illustrates the fan of a male wildtype strain N2.

Acknowledgments

The authors thank Federico Belloni for initiating the HOT project, Sophie Ugolini and Eric Vivier for general discussion and advice, Helmut Wurm, Peter Waltinger and Gunter Köhn for their advice and technical assistance, Nassima Benmansour for providing T cells and all members of the two teams for constant support. Jonathan Hodgkin for providing the bus mutants and the Caenorhabditis Genetics Center for the wild type strain, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

Supporting Material

An additional materials and methods section, five figures and seven movies are available at www.biophys.org.

Grants

Supporting Citations

References [START_REF] Allen | Differential requirements for antigen processing by macrophages for lysozyme-specific T cell hybridomas[END_REF][START_REF] Babbitt | Antigenic competition at the level of peptide-Ia binding[END_REF]

SUPPORTING MATERIALS AND METHODS

Cell culture mCD4 3A9 T cells (1,2) were grown in RPMI (Gibco, Life technologies, Saint-Aubin, France) supplemented with 5 % heat-inactivated fetal bovine serum (Gibco), sodium pyruvate 1 mM (Gibco) and HEPES 10 mM (Gibco) at 5 %CO2 and 37°C in a humidified atmosphere.

Labeling method

For cell labeling, mCD4 3A9 T cells (10 5 in 100 μl) were incubated in presence of FITC-labelled mAb anti-CD45 (Becton Dickinson Company, Le Pont-de-Claix, France) or PE-labelled mAb anti-CD45 (Becton Dickinson) at final concentration of 0.5 μg/μL for 10 min at 4°C. Cells were washed three times by centrifugation (3 min at 200 × g) in complete medium. Cells were imaged in HBSS (Gibco) supplemented with 10 mM HEPES (Gibco) in Lab-Tek well chamber (Thermo scientific, Dominique Dutscher, Brumath, France). Acquisition was performed using 40 focal plane separated by 500 nm, with 200 ms exposure time.

SUPPORTING FIGURES

Figure S1 -Observation chamber designed for controlled C. elegans infection experiments

A two well chamber slide linked by a small channel has been designed with a reservoir for the spores and the other for the tests of adhesion. Wells were fabricated in an aluminum piece. The bottom face was covered by a double-sided tape in which an incision makes the channel. A size #1 cover glass seals the chamber.

LEGENDS OF THE SUPPORTING MOVIES

Movie S1 -3D image of a bead with or without synchronization

A single latex bead (red dot) from the ones settled down on the cover glass is trapped and imaged in brightfield (stack of 10 images at 1 ȝm intervals). Sample was moved by the z-top plate without synchronization (left panel), or with the trapped bead maintained in its initial position by synchronization (right panel). The z position of the image focal plane is specified in the bottom left corner of the movie. In the right panel, the persistency of the trap is demonstrated by controlled displacement of the bead to the left.

Movie S2 -3D image of a cell with or without synchronization

A membrane-stained T cell hybridoma (CD4 3A9 T cell) was trapped and observed by confocal imaging (stack of 20 images at 1 ȝm intervals). Cell was imaged with OFF or ON synchronization between trap and imaging, left and right panels, respectively. The z position of the image focal plane is specified in the bottom left corner of the movie.

Movie S3 -Independent 3D displacement of fluorescent spores D. coniospora spores were stained by DiI. The movie illustrates the confocal images recording the independent motion of five spores in xyz direction.

Movie S4 -Adhesion test performed by displacement of a sedated worm

The movie illustrates confocal imaging recorded during an adhesion test in which a sedated worm is moved by the xyz stage of the microscope to contact in head area three stationary trapped spores. D. coniospora spores are stained by DiI. The C. elegans strain expressed the GFP in the pharynx and epidermis.

Movie S5 -Adhesion test performed by displacement of multiple independent trapped spores

The movie illustrates confocal imaging recorded during an adhesion test in which three trapped spores are moved to contact one at a time the head area of a worm: the first spore instantly adheres but disappears from the focal plan (it is still adherent as illustrated at the end of the movie recording defocused images); the second one adheres also immediately to the head and stays in the focal plane; the third one becomes adherent after two consecutive contacts. This is mainly due to a defective orientation of the conidium part of the spore contacting the cuticle surface. D. coniospora spores are stained by DiI. The C. elegans strain expressed the GFP in the pharynx and epidermis.

Movie S6 -3D multicolor confocal imaging during adhesion of a single spore on C. elegans

The movie illustrates 3D confocal images in three colors recorded as in Fig. 5, during and after the adhesion of a spore on the head area of a worm. D. coniospora spores are stained by DiI. The C. elegans strain expressed the GFP in the epidermis (green) and the RFP in the pharynx (yellow). Its cuticle and neurons are stained by DiD (red).

Movie S7 -3D multicolor confocal imaging of T cells moved by HOT

Two subsets of mCD4 3A9 T cells were stained by mAb anti-CD45 labeled by FITC or PE and then mixed together. Cells were trapped and moved in 3D independently from each other.