He ´lène Pichot
email: helene.pichot@ass.nc

Bruno Pereira

Elodie Magnat

Martine Hennequin

Ste ´phanie Tubert-Jeannin

Implementation and impact of a dental preventive intervention conducted within a health promotion program on health inequalities: A retrospective study

Background

The objective of this retrospective survey was to evaluate after one year, the conditions and impacts of a dental sealant intervention conducted in New Caledonia, within a health promotion program. A greater or at least equivalent quality and impact of the intervention was expected for children living in socially deprived regions with the greatest health needs.

Methods

The study population was the schoolchildren, aged 6 years in 2016, who benefited from the dental sealant program (n = 2532). The study sample was randomly selected in 2017 from that population (n = 550). The children's dental status was evaluated at school in 2017 and compared with that recorded in 2016 during the sealant intervention allowing the calculation of the retention rates and one-year carious increment on first permanent molars. Sociodemographic variables (gender, public/private school) and conditions of sealant placement (school/dental office, presence of a dental assistant) were recorded. The carious increment was explained using a mixed multiple random-effects regression. A mediation analysis was conducted to assess the respective contributions of the retention rates and the region of origin on caries increment.

Results

The participation rate was very high (89%) and on average, children had 83% of their dental sealants present after one year, 31% fully and 52% partially present. Caries increment varied depending on the sealant retention rate as well as on the region (North, South, Islands).

Introduction

Oral diseases remain a major health problem in both developing and developed countries [START_REF]Global Burden of Disease Study 2013 Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013[END_REF]. Indeed, the prevalence of untreated oral conditions is high worldwide. Low-income and socially-or medically-disadvantaged populations experience higher rates of chronic diseases and this gradient is particularly apparent for oral diseases [START_REF] Marmot | Understanding social inequalities in health[END_REF].

Since oral health inequalities come across the whole social body according to a gradient, the challenge is to take into account that gradient when evaluating public health interventions. An efficient intervention might have no effect on health inequalities if all socioeconomic groups benefit equally. And it may even increase health inequalities if the wealthiest groups benefit more [START_REF] Lorenc | What types of interventions generate inequalities? Evidence from systematic reviews[END_REF]. Indeed, the more affluent communities are often in a better position to implement health promotion programs not only for financial reasons but also because of environmental factors or manpower resources. Unfortunately, there is very little evidence concerning the equity effects of health-related interventions. Thus, studies assessing the impact of universal interventions on children with various social or environmental backgrounds are needed to identify the differential effects as well as the impact of the implementation conditions [START_REF] De Silva | Community-based population-level interventions for promoting child oral health[END_REF].

New Caledonia (NC) (245,580 inhabitants) is a French Oceanian overseas territory, with extensive administrative autonomy. The population is a mix of 40% Kanak (indigenous population of New Caledonia), 30% White European (Caledonians and Metropolitans), 10% Polynesian (Wallisians) and South-East Asian or Vanuatu people. The population varies depending on the region, with more Kanak in the North and the Islands and more Polynesians, white Europeans and Asians in the South. Three quarters of the population live in the South, where the economic activity is concentrated, and 39% of the population lives in Noume ´a city. New Caledonia is one of the richest countries in the South Pacific area but there are strong social and economic disparities between regions (North, Islands, South) [START_REF]Recensement de la population en Nouvelle-Cale ´donie en[END_REF]. As an example, the proportion of households having a computer at home varied in 2014 from 37% in the Islands, to 53% in the North and 76% in the South.

A study conducted in 2011-2012 has evaluated the dental and weight status of 6-, 9-and 12-yr-old children in NC. The prevalence of untreated dental caries was almost 60% among 6and 9-yr-olds and about 50% among 12-yr-olds. Caries experience was unevenly distributed in the population, with one third of 12-yr-olds having more than five untreated carious teeth. The number of carious lesions was related to their unfavourable lifestyle, deprived social status and lack of preventive dental care [START_REF] Pichot | Dental status of new caledonian children: is there a need for a new oral health promotion programme?[END_REF]. The prevalence of overweight and obesity was high and greatly increasing between the ages of 6 (respectively 10.8% and 7.8%) and 12 (respectively 22.2% and 20.5%), with one third of the 12-yr-olds having an excess of abdominal adiposity. Geographical location (region), ethnicity, tooth-brushing frequency and masticatory function were significant risk factors for oral diseases and overweight/obesity [START_REF] Tubert-Jeannin | Common risk indicators for oral diseases and obesity in 12-year-olds: a South Pacific cross sectional study[END_REF]. People with the lowest levels of resources also showed difficulties attending for dental care despite the public medical/ dental coverage existing in NC.

That study highlighted the need for new strategies aimed at improving children's oral and general health and at reducing inequalities. An alternative approach to traditional healthcare was thus chosen in collaboration with NC local populations and government, based on the principles of Oral Health Promotion (OHP) [START_REF] Fry | Applying the Ottawa Charter to inform health promotion programme design[END_REF] with the development of-1-health education in schools in order to promote the adoption of health promoting behaviours [START_REF] Langford | The World Health Organization's Health Promoting Schools framework: a Cochrane systematic review and meta-analysis[END_REF], 2-preventive interventions, such as the implementation of tooth brushing at school, for the child population, 3-a reorientation of dental services towards more effective and preventive interventions. The OHP program ("Mes dents, Ma sante ´") was developed in connection with other health programs related to the prevention of other chronic diseases such as rheumatic heart disease or obesity. The common risk factor approach was thus privileged in order to address risk factors common to those chronic conditions within the context of the local environment [START_REF] Watt | Integrating the common risk factor approach into a social determinants framework[END_REF].

Within this framework, the dental sealant program, which has existed since 2009, was renewed in order to ensure its quality and that all 6-yr-old schoolchildren can benefit from it. Indeed, dental sealants have been shown to be an effective preventive intervention when evaluated in real-life school conditions [START_REF] Ahovuo-Saloranta | Pit and fissure sealants for preventing dental decay in permanent teeth[END_REF][START_REF] Muller-Bolla | Effectiveness of school-based dental sealant programs among children from low-income backgrounds in France: a pragmatic randomized clinical trial[END_REF]. The program is now coordinated by the NC health agency in connection with regional educational and health authorities, health-fund bodies and the dental profession. A training course and a standardized protocol were implemented in 2014 in order to ensure the quality and relevance of sealant applications by dentists, in schools or public dental services.

The objective of this retrospective study was to evaluate after one year, the dental sealant program conducted in 2016 among 6-yr-old children in NC, by measuring the participation and retention rates [START_REF] Muller-Bolla | Retention of resin-based pit and fissure sealants: A systematic review[END_REF]. The aim was also to evaluate the influence of the quality of the intervention (retention rates) on the one-year caries increment, while taking into account initial dental status, conditions of sealant placement and social determinants such as the region of origin. The objectives of this study are positioned within the general aim of taking into account oral health inequalities when assessing public health interventions. Indeed, to ensure that public health interventions do not increase health inequalities, a greater or at least equivalent quality and health outcome of those interventions are expected in socially deprived sectors with greatest health needs compared to more favoured areas [START_REF] Lorenc | What types of interventions generate inequalities? Evidence from systematic reviews[END_REF].

Population and methods

Population and study sample

The study population was the group of schoolchildren aged 6 in 2016 who benefited from the dental sealant program; the program concerned 4329 children, was effectively offered to 3774 (87%) children (351 in the Islands, 908 in the North and 2515 in the South); 2532 children participated (89% in NC, 92% in the Islands, 88% in the North and 89% in the South).

The number of children to be selected in the 2017 study from that population was calculated (n = 500) to ensure the precision of the retention rates' estimates (rate = 50%, precision 5%) and to highlight an effect size above 0.5 when considering variations between regions. This number was also calculated following the criteria used in multidimensional and mediation analyses [START_REF] Cohen | Statistical Power Analysis for the Behavioral Sciences[END_REF][START_REF] Hair | Multivariate Data Analysis[END_REF][START_REF] Fritz | Required sample size to detect the mediated effect[END_REF]. Children were randomly selected using a computerised, clustered sampling method with a probability proportional to the regions' population.

Ethics approval and consent to participate

Study approvals were obtained from the NC educational and health institutions. Schools were approached through local educational authorities. Data were recorded in a file registered within the French ethical committee for data management (CNIL -Commission Nationale Informatique et Liberte ´, No. kpP1390145R). Information letters and consent forms were sent to the parents. The children whose parents returned written consent were examined. In order to prevent negative impact on the participation rate in the OHP program, it was decided not to collect individual social information or behavioural data at this stage.

Fissure sealant program

The age of 6 was chosen because school is compulsory from this age and it allowed the sealant to be applied on first permanent molars as soon as they erupted. The recommendation for the dentists is to seal all non-carious or with only very early enamel lesions (ICDAS 1 ) erupted first permanent molars whatever the caries risk [START_REF]International Caries Detection and Assessment System (ICDAS) Coordinating Committee. International Caries Detection and Assessment System (ICDAS II)[END_REF]. Caries risk is very high in NC and it was considered not feasible nor pertinent to differentiate children according to caries risk. Photopolymerised resin sealants (3M Cleanpro) were applied with cotton rolls for saliva isolation [START_REF] Siegal | Targeting school-based dental sealant programs: who is at "higher risk[END_REF][START_REF] Simonsen | A review of the clinical application and performance of pit and fissure sealants[END_REF]. Sealants were applied either at school in a mobile dental surgery or in public dental offices. Thirty general dental practitioners performed the 2016 dental examinations and applied the fissure sealants. They were recruited by the NC health agency within public regional health services and through the council of private dentists. The practitioners were assigned to one or several schools, depending on feasibility criteria that were mostly dependent on geographical locations. Children from the same school were examined by the same practitioner. Prior to the start of the program, all the practitioners underwent a training course consisting of a presentation of the NC OHP program, the sealant application protocol, illustrations with clinical situations and training about the data collection process.

Study variables

The children's dental status was evaluated in 2017 at school. Radiographs were not used. Six general dental practitioners performed the children's examinations, after having been calibrated through a training course described in a previous study [START_REF] Pichot | Dental status of new caledonian children: is there a need for a new oral health promotion programme?[END_REF]. The presence of dental sealants was recorded. A sealant was considered as fully or partially present using standardised criteria [START_REF] Simonsen | A review of the clinical application and performance of pit and fissure sealants[END_REF]. For each child, the number of teeth with partially remaining sealant and fullyintact sealant was recorded. The clinical information collected in 2017 was connected to the 2016 individual data. The number of sealants applied in 2016 was compared with the number of sealants present during the 2017 examination. This allowed the calculation of three retention rates for each child; the complete retention rate (based on fully-present sealants), the partial retention rate (partially present) and the global retention rate (fully or partially present).

Dental caries were diagnosed at the dentinal threshold level (ICDAS 4-6 ) for deciduous and permanent teeth [START_REF]WHO | Oral health surveys: basic methods -5th edition[END_REF]. The dental status (DT first molars : number of carious first permanent molars) recorded in 2016 during the sealant intervention was compared with that of the 2017 dental examination. This allowed the calculation of the one-year carious increment: ΔDT 1st molars = DT first molars in 2017-DT first molars in 2016.

Socio-demographic variables (gender, region, public or private school) were recorded from the school registers. The setting where dental sealants were applied in 2016 (school vs dental office) and the presence of a dental assistant during the sealing procedure were also retrieved for each child.

Data analysis

Data errors were corrected before analysis. The statistical analyses were performed using the Stata statistical software package, version 13.

Retention rates were expressed with 95% confidence intervals (CI). The influence of initial dental status, conditions of sealant placement and the child's profile (gender, school, region) was explored using bivariate analysis.

Variations in the distribution of the study variables (retention rates, initial and 2017 dental status, condition of sealant placement, sociodemographic profile, caries increment) depending on the region (an ecological variable representing a major social determinant in NC) were also explored using bivariate analysis. Chi-squared, ANOVA or Student t-tests were used depending on the variables being considered. For the main variables, when p-value was significant, a Tukey-Kramer post-hoc test was performed to take into account multiple comparisons.

A mixed multiple random-effects regression was used to evaluate the influence of the quality of the intervention (retention rates), initial dental status, conditions of sealant placement and the region of origin on the one-year caries increment. Mixed models were used including the examiner parameter as random effect [START_REF] Snijders | Multilevel Analysis: An Introduction To Basic And Advanced Multilevel Modeling[END_REF][START_REF] Verbeke | Linear Mixed Models for Longitudinal Data[END_REF][START_REF] Hox | Multilevel Analysis: Techniques and Applications[END_REF][START_REF] Goldstein | Multilevel Statistical Models[END_REF]. The caries increment was not normally distributed (even with a normality transformation), and thus the 0-inflated Poisson model was used. The interactions between factors were tested.

We conducted a mediation analysis to assess the respective contributions of the retention rate and the region on caries increment. A mediation proportion was estimated, indicating how much of the whole increment value provided by an independent variable (the region) can be explained by the indirect path in which changes in this independent variable drives a change in the mediator (retention rate), and changes in the mediator then affect outcome [START_REF] Imai | Unpacking the Black Box of Causality: Learning about Causal Mechanisms from Experimental and Observational Studies[END_REF][START_REF] Shrout | Mediation in experimental and nonexperimental studies: new procedures and recommendations[END_REF]. We performed multilevel mediation analysis with other explanatory variables and examiner effect being integrated. Three models were conducted depending on the region being considered as the reference (A-South, B-Islands, C-North). The global and complete retention rates were considered separately. Results were summarized using a graph giving the mediation proportion and significance of the mediation analysis associations.

Results

Of the children selected in the 2017 sample, 459 children participated and were examined. Among the participants, 48% were girls, 84% attended public schools, 9% lived in the Islands, 29% in the North and 62% in the South.

On average, children still had 83% (95%CI: 79.9%-86.2%) of their dental sealants after one year; 31.1% (27.8%-34.5%) were still fully present and 51.9% (48.6%-55.1%) were partially present. Retention rates did not vary depending on the children gender, type of school (private vs public) or the conditions of sealant placement (dental office vs school, presence of a dental assistant). Dental status in 2016, as well as the number of sealants applied in 2016, were not associated with retention rates. Retention rates appeared to be slightly better in the North and lower in the South (Table 1 andFig 1).

In 2016, the mean number of sealed teeth was 3.34 (1.01) with 39 children (8%) having had one tooth sealed and 295 (64%) four teeth sealed. It was found that 31% of the children benefited from the sealant program within schools and a dental assistant was present in 84.9% of the cases.

The South showed a very high rate of sealant application at school with an assistant. In 2016, caries prevalence was high with non-treated carious lesions in eight children out of ten in the Islands (Table 1). In 2017, all children had their 4 first permanent molars erupted with no filling being recorded. It was observed that 63.6% of the children (58.6%-67.7%) had untreated carious lesions on permanent or temporary teeth. The 2017 caries prevalence varied depending on the region. In total, 168 first molars had carious lesions with a mean number of decayed teeth per child of 0.35 (0.84). The mean number of sealants on first permanent molars was 2.82 (1.26) with only 25 children having no sealants and 43% with all their molars sealed.

Carious increment ΔDT 1st molars was 0.18 (0.76) and varied depending on the region with a much higher caries increment in the Islands (Table 1). The multidimensional model showed that caries increment varied significantly depending on the global sealant retention rate as well as the region (Table 2). Children living in the South and North and children with a high retention rate experienced less new carious lesions. The children's sociodemographic profile, modalities of sealant application as well as initial dental status were not significant factors. There was a significant examiner effect that was managed by using a mixed multiple randomeffects regression. The same findings were nearly obtained when considering the complete retention rate (Regression Coefficient: -0.54, 95% CI: -0.74; -0.34, p<0.0001).

A multilevel mediation analysis was performed to evaluate if the retention rate was a mediator of the effect of the region on caries increment. The direct association between the region and caries increment remained significant only in Model B. In models A and C, the retention rate was linked to the region. The modalities of sealant application significantly influenced that relationship for the three models. For Model B, the influence of the region increased when retention rate was considered as a mediator. This means that living in the Islands was a strong determinant for high caries increment particularly when the retention rate was low. For Models A and C, the retention rate was not a mediator for the relationship between the region and caries increment. The mediator effect was lower for the complete retention rate (35%) as compared to the global retention rate (38%) (Fig 2).

Discussion

The objective of this retrospective study was to evaluate the quality and the impact of the dental sealant program on oral health inequalities. Findings showed that the participation rate was very high and that on average, children had approximately 80% of their dental sealants present after one year, 30% being fully present and 50% partially present. The aim was also to evaluate Step 1: The first step in our mediational analysis was the finding that belonging to the Islands region as compared to the North and South regions had a measurable impact on caries increment after accounting for baseline risk covariates.

Step 2: Second, we checked if the retention rate (mediator) was related with the region, after accounting for baseline risk covariates.

Step 3: Finally, a multilinear regression (mixed model) calculated the influence of the region on the tested mediator (retention rate). Subsequently, we jointly calculated the influence of the mediator and the direct effect of the independent variable on caries increment after accounting for baseline risk covariates. This last step shows that retention rate partially mediates [38%. P = for the average causal mediation effect (ACME)] the original effect of the region on caries increment and consequently remains directly associated with caries increment in an independent manner. The mediator (retention rate) and the main independent variable (Region) are assessed as binary variables.

https://doi.org/10.1371/journal.pone.0230639.g002

the impact of retention rates, social determinants and other explanatory factors on caries increment for first permanent molars. Caries increment varied depending on the retention rates as well as on the region. The mediation analysis showed that living in a more deprived area was a strong determinant for high caries increment particularly when the retention rates were low. The notion of retention is crucial because the main function of sealants is to form an efficient physical barrier between the enamel surface and the oral environment. Thus, retention rates are used as clinical evaluation criteria to measure effectiveness. The mean retention rate in this study is comparable to global retention rates found in other pragmatic studies that have evaluated the school sealant program [START_REF] Dorantes | Assessment of retention rates and clinical benefits of a community sealant program[END_REF][START_REF] Venker | Twelve-month sealant retention in a school-based program using a self-etching primer/adhesive[END_REF][START_REF] Ismail | An evaluation of the Saskatchewan pit and fissure sealant program: a longitudinal followup[END_REF]. However, the mean complete retention rate observed here (30%) was relatively low as compared to other studies with much higher rates (50-70%) [START_REF] Muller-Bolla | Retention of resin-based pit and fissure sealants: A systematic review[END_REF][START_REF] Williams | An evaluation of a school-based dental sealant programme[END_REF][START_REF] Gugnani | Trial shows caries reductions at one year in school-based sealant programme[END_REF][START_REF] Heifetz | Retention of a fluoride-releasing sealant compared with its non-fluoride analogue: interim results of a clinical study after an average of eight months[END_REF][START_REF] Francis | Retention and effectiveness of fissure sealants in Kuwaiti school children[END_REF]. The high proportion of partially present dental sealants could lower the effectiveness of the program in preventing dental caries development [START_REF] Tianviwat | Loss of sealant retention and subsequent caries development[END_REF]. This low rate could be explained by the age of the children whose first molars were erupting at the time of sealant placement or by the fact that all teeth were sealed and not only teeth at high risk of caries. These rates might also be related to the dentists' lack of involvement in preventive activities. The NC sealant program has been conducted within the French context where the population benefits from Universal Health/Dental Coverage. But dentists are mainly working in private practices with no (or few in NC) community dental services or the presence of associated professionals such as dental hygienists [START_REF] Pegon-Machat | The healthcare system and the provision of oral healthcare in EU Member States: France[END_REF]. Historically, priority has been given to operative care, at the expense of preventive dentistry. Many preventive interventions are not covered by the National Health Insurance system. Dentists thus tend to focus on restorative treatments and their management of carious disease is mainly invasive and curatively driven [START_REF] Mazevet | The highly controversial payment reform of dentists in France: Seeking a new compromise after the 2017 strike[END_REF].

The dental sealant intervention is integrated within an oral health promotion plan. Evidence of the effect of educational interventions or multi-component school-and communitybased interventions is equivocal [START_REF] Lorenc | What types of interventions generate inequalities? Evidence from systematic reviews[END_REF]. Thus, the evaluation of the impact of the program according to the social situation and oral health needs is very important as public health interventions may increase inequalities in the population. The 'inverse care law' states that those most in need of benefiting from preventive interventions are the least likely to receive and benefit from them. Some interventions are successful at improving health across the population but they may increase health inequalities. This can happen when an intervention is of greater benefit to advantaged (lower-risk) groups than to disadvantaged (higher risk) groups [START_REF] Winter | Long-term effect of intensive prevention on dental health of primary school children by socioeconomic status[END_REF][START_REF] Ripa | Caries prevalence, treatment level, and sealant use related to school lunch program participation[END_REF]. Conversely, some interventions may reduce inequalities, if they are of greater benefit to disadvantaged groups. In this study, it was demonstrated that the participation rate was very high whatever the region and that the setting of fissure sealant placement (school vs public dental offices) did not influence the retention rates. The good balance of participation and quality of intervention has been obtained by focusing efforts on ensuring accessibility to the intervention in every place, even the more deprived and isolated ones. In the Islands, caries prevalence was high in 2016 and caries increment was also marked between 2016 and 2017. The mediation analysis showed that having a high retention rate was particularly important for the children in the Islands. Hence, the retention rate was a good intermediate indicator of the effect of the preventive intervention on oral health inequalities, showing that accessible and high-quality interventions are crucial for deprived populations. This is an illustration of the need for proportionate universalism in oral health promotion programs with reinforced resources adapted to the needs of the populations in deprived sectors.

These findings were obtained within a short follow-up period; the calculation of a two-year (or more) caries increment might have led to the measurements of higher impacts. Moreover, it was not possible to make a comparison with a control group as all 6-yr-old children are concerned by the program in NC. Since the Ottawa declaration [START_REF] Fry | Applying the Ottawa Charter to inform health promotion programme design[END_REF], there has been an expansion of health promotion programs but there is still a need for an evidence-based evaluation of their impact. Randomized Clinical Trials are needed but also other types of studies such as pragmatic studies conducted in real life contexts. Evaluation studies have to be context sensitive to identify what works for whom and to take into account the implementation processes, and the barriers and facilitators to health promotion programs [START_REF] Walach | Circular instead of hierarchical: methodological principles for the evaluation of complex interventions[END_REF]. Health promotion programs are frequently conducted in school environments or in available community settings. Understanding local contexts as socially-complex systems may help adopt better suited approaches for the dissemination and evaluation of oral health promotion programs [START_REF] Keshavarz | Schools as social complex adaptive systems: a new way to understand the challenges of introducing the health promoting schools concept[END_REF]. In the present study, sealant application procedures and evaluation process were adapted to the local situation. This has limited the quality of the program and evaluation protocol but, at the same time, this study provided invaluable context-sensitive data that helped understand what works and for whom within a pragmatic perspective.

The oral health promotion program (OHP) conducted in NC since 2014 was developed after the identification of oral health needs, but also after their recognition by the population and major local stakeholders [START_REF] De | Le plan Do Kamo en 86 actions[END_REF]. The sealant application program that is part of the OHP program has been put in place within the context of routine practice. Existing intervention practices were identified and improvements were facilitated. The sealant intervention has been supported first because it is an evidence-based intervention with a solid rationale. Moreover, a user-centred approach was privileged and an interdisciplinary team of researchers, political stakeholders together with health providers were involved in the program. The sealant program was thus integrated in the OHP program as the likelihood of adoption and implementation in daily practice was high.

The sealant program was associated with other interventions aimed at modifying other health determinants such as developing a health education program or promoting tooth brushing in nurseries (age 3-6 years) and schools [START_REF] Macpherson | Childsmile: the national child oral health improvement programme in Scotland. Part 1: Establishment and development[END_REF]. These interventions will be evaluated progressively as soon as their implementation has been completed [START_REF] Bleijenberg | Increasing value and reducing waste by optimizing the development of complex interventions: Enriching the development phase of the Medical Research Council (MRC) Framework[END_REF]. It must be stated that the level of implementation of tooth brushing at school is very promising (85%) with a high involvement of schools in the deprived areas such as the North region where community workers and teachers became highly involved (https://www.ass.nc). An epidemiological survey was conducted in 2019, among a national representative sample of 6-, 9-and 12-yr-old children. This survey will bring more information about the evolution of children's oral health in NC over the last 5 years and the effect of the OHP program.

Conclusions

This retrospective study of the quality and impact of the fissure sealant program is part of the evaluation process of a larger OHP program. This study showed a high participation rate and acceptable effectiveness as measured with the one-year retention rates, for a fissure sealant intervention conducted in real-life conditions and integrated in a large health promotion program. Nevertheless, even with well-balanced participation and retention rates between regions, the intervention was not effective enough to totally balance the influence of health determinants, especially in socially-deprived sectors characterized by greater dental needs. These results pointed out the issues of generalizing well-proven effective preventive procedures within specific real-life contexts. The results emphasised the need for continuous and targeted training programs for the program actors to help optimize effectiveness. It would have been illusory to think it possible to counterbalance the effects of social determinants on health with one short-term preventive intervention, even if integrated in a larger health promotion program. Nevertheless, the results show that taking into account the social gradient of health when evaluating public health interventions is a major issue. Further studies to evaluate long-term participation, quality and effects of the whole health promotion and education program in NC are needed, as well as cost-effectiveness studies to help support sound future public health decisions.

Fig 1 .

 1 Fig 1. Complete and global retention rates and caries increment (ΔDT 1st molars ) depending on the region and setting of sealant placement. https://doi.org/10.1371/journal.pone.0230639.g001

Fig 2 .

 2 Fig 2. Mediation analysis of caries increment (Model B).Tested mediator: Global retention rate. Independent variable: Region (Model B: the Islands compared to the North and South). Baseline risk co-variates: gender, type of school (private vs public), dental status in 2016 (number of first molars and % with carious lesions), conditions of sealant placement (school vs dental office, with or without dental assistant), examiner effect (as random effect). Step 1: The first step in our mediational analysis was the finding that belonging to the Islands region as compared to the North and South regions had a measurable impact on caries increment after accounting for baseline risk covariates. Step 2: Second, we checked if the retention rate (mediator) was related with the region, after accounting for baseline risk covariates. Step 3: Finally, a multilinear regression (mixed model) calculated the influence of the region on the tested mediator (retention rate). Subsequently, we jointly calculated the influence of the mediator and the direct effect of the independent variable on caries increment after accounting for baseline risk covariates. This last step shows that retention rate partially mediates [38%. P = for the average causal mediation effect (ACME)] the original effect of the region on caries increment and consequently remains directly associated with caries increment in an independent manner. The mediator (retention rate) and the main independent variable (Region) are assessed as binary variables.

Table 1 . Description of the study variables, for the whole sample and per region.

 1

		Total n = 459	North n = 132	Islands n = 43	South n = 284	P
	RETENTION RATES
	Partial retention rate	0.52 (0.35) �	0.59 (0.38)	0.45 (0.32)	0.50 (0.34)	0.004!
	Complete retention rate	0.31 (0.36)	0.30 (0.36)	0.42 (0.37)	0.30 (0.36)	NS
	Global retention rate	0.83 (0.35)	0.89 (0.31)	0.87 (0.45)	0.80 (0.34)	0.02!
	CONDITIONS: SEALANT APPLICATION
	% of sealants placed at school	69.1%	44.7%	25.6%	87.0%	<0.001
	Presence of a dental assistant (n = 444)	84.9%	64.7%	73.2%	95.1%	<0.001
	SOCIODEMOGRAPHIC PROFILE
	% in private schools	15.9%	19.7%	41.9%	10.2%	P<0001
	% of girls	48.0%	40.2%	55.8%	50.4%	NS
	INITIAL DENTAL STATUS
	Number of First molars present in 2016	3.63 (0.81)	3.64 (0.76)	3.47 (0.88)	3.64 (0.82)	NS
	% of children with caries in 2016 £	63.6%	69.7%	79.0%	58.5%	0.007
	Number of carious first molars in 2016	0.19 (0.54)	0.20 (0.57)	0.14 (0.41)	0.19 (0.53)	NS
	Number of sealants applied in 2016	3.34 (1.01)	3.41 (0.92)	3.21 (1.06)	3.32 (1.03)	NS
	2017 DENTAL STATUS
	Number of carious first molars in 2017	0.35 (0.84)	0.32 (0.81)	0.65 (0.90)	0.37 (0.84)	0.06
	Number of sealants remaining in 2017	2.82 (1.26)	3.08 (1.23)	2.74 (1.36)	2.72 (1.25)	0.03
	% of children with caries in 2017 £	63.0%	68.9%	88.4%	56.4%	<0.001
	ONE-YEAR CARIES INCREMENT
	Caries increment: ΔDT 1st molars	0.18 (0.76)	0.12 (0.59)	0.51 (0.83)	0.15 (0.81)	<0.001 §
	n: number of subjects,

� mean (standard deviation) for the proportion of sealants remaining per child, p: p value for Chi square test or ANOVA. £ permanent and temporary teeth, ! the South region differed from the North region (Tukey-Cramer). § the Islands differed from the North and from the South (Tukey-Cramer). https://doi.org/10.1371/journal.pone.0230639.t001

Table 2 . Significant regression coefficients derived from the mixed multiple random-effects regression with one- year carious increment (ΔDT 1st molars ) as dependent variable.

 2

		ΔDT 1st molars
	RC	95% CI	p

North, South), dental status in 2016 (number of first molars and % with carious lesions), conditions of sealant placement (school vs dental office, with or without dental assistant), examiner effect, p = 0.002 https://doi.org/10.1371/journal.pone.0230639.t002

PLOS ONE | https://doi.org/10.1371/journal.pone.0230639March 24, 2020

, Untreated carious lesions recorded when dentine is involved; DT 1st molars , Number of untreated carious lesions on first permanent molars; ΔDT 1st molars , One-year caries increment for first permanent molars; CNIL, Comite ´National Informatique et Liberte ´(French ethical committee for data management).

Acknowledgments

The authors would like to thank Caroline Eschevins (CROC, Dental School, Clermont-Ferrand, France) for her help in the writing of the manuscript.

The author(s) received no specific funding for this work

The data underlying this study are available from Zenodo using DOI: 10. 5281/zenodo.3670886.


Marion Millerand

Laure Sudre

Meriam Nefla

F Pène

Chloé Rousseau

Anna Pons

Christophe Rousseau

Arnaud Ravat

Shizuo Akira

Takashi Satoh

Dr Francis Berenbaum
email: francis.berenbaum@sat.aphp.fr

Claire Jacques

: Umr_S938

Activation of innate immunity by 14-3-3 ϵ, a new potential alarmin in osteoarthritis

Keywords: 14-3-3 ε, alarmin, osteoarthritis, Innate immunity, TLR, synovitis Running headline : 14-3-3 marrow derived macrophages BSA: bovine serum albumin cDNA: complementary deoxyribonucleic acid DAMP: damage-associated molecular pattern monocyte chemoattractant protein-1 MMP: matrix metalloproteinases mRNA: messenger ribonucleic acid OA: osteoarthritis OxPAPC: Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-

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

Introduction

Osteoarthritis (OA) is a highly complex and the most prevalent joint disorder with a total of 242 million people affected worldwide [START_REF]Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013[END_REF] . Clinical symptoms include severe pain, joint stiffness and reduced function, which seriously decrease quality of life [START_REF]Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013[END_REF] . Unfortunately, only a few drugs are weakly effective for treating symptoms, and no disease-modifying osteoarthritis drugs (DMOADs) are available to date. Initially considered cartilage driven, OA is a much more complex disease with inflammatory mediators released by cartilage, bone and synovium [START_REF] Felson | Osteoarthritis of the Knee[END_REF] . Increasing evidence suggests that inflammation is present in OA and has raised the possibility that inflammation and the innate immune system could be active players in the development and progression of OA [START_REF] Hügle | What drives osteoarthritis?-synovial versus subchondral bone pathology[END_REF][START_REF] Berenbaum | Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!)[END_REF] . Innate immune responses involve the activation of resident leukocytes such as macrophages, production of inflammatory mediators (cytokines, chemokines, and lipid metabolites), recruitment of neutrophils and monocytes/macrophages, and aims to eliminate invading microorganisms and injured tissues [START_REF] Herrero-Beaumont | Targeting chronic innate inflammatory pathways, the main road to prevention of osteoarthritis progression[END_REF] . Histopathological studies have confirmed that immune cell infiltration is extremely common in OA histological specimens [START_REF] Chen | Sterile inflammation: sensing and reacting to damage[END_REF] . The innate immune system participates in inflammation triggered by host molecules or fragments collectively called DAMPS (danger-associated molecular patterns) or alarmins [START_REF] Herrero-Beaumont | Targeting chronic innate inflammatory pathways, the main road to prevention of osteoarthritis progression[END_REF][START_REF] Furman | Articular Ankle Fracture Results in Increased Synovitis, Synovial Macrophage Infiltration, and Synovial Fluid Concentrations of Inflammatory Cytokines and Chemokines[END_REF][START_REF] Oppenheim | Alarmins: chemotactic activators of immune responses[END_REF] .

The term "alarmin" was proposed by Oppenheim and co-workers in 2005 to classify proteins that are rapidly released during infection or tissue damage, activating immune cells after interaction with their specific receptors [START_REF] Furman | Articular Ankle Fracture Results in Increased Synovitis, Synovial Macrophage Infiltration, and Synovial Fluid Concentrations of Inflammatory Cytokines and Chemokines[END_REF] . Alarmins are now considered to be markers of destructive processes in joints [START_REF] Nefla | The danger from within: alarmins in arthritis[END_REF] . Because of their fast release as a result of cell stressor nonprogrammed cell death, alarmins are among the first factors to be secreted and, as such, act as first responders to stimuli.

In addition to their role in disease initiation, alarmins also amplify and sustain inflammatory processes and, thus, play a notable role in the pathogenesis of inflammatory conditions [START_REF] Liu-Bryan | Emerging regulators of the inflammatory process in osteoarthritis[END_REF][START_REF] Manfredi | Regulation of dendritic-and T-cell fate by injury-associated endogenous signals[END_REF] . A number of alarmins have been detected at high levels in OA tissues and synovial fluid, including HMGB1, UA, ATP, thymosin ß4 and various S100 proteins, enhancing catabolic processes and inflammatory responses that contribute to disease progression [START_REF] Oppenheim | Alarmins: chemotactic activators of immune responses[END_REF][START_REF] Tamaki | Expression of Toll-like Receptors and Their Signaling Pathways in Rheumatoid Synovitis[END_REF][START_REF] Ke | Synovial Fluid HMGB-1 Levels are Associated with Osteoarthritis Severity[END_REF][START_REF] Wang | S100A12 levels in synovial fluid may reflect clinical severity in patients with primary knee osteoarthritis[END_REF] .

Synovitis, the inflammation of the synovium, can occur in early stages of OA [START_REF] Sunahori | The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis[END_REF] .

OA synovitis directly contributes to several clinical signs and symptoms including joint swelling and effusion, and reflects the structural progression of the disease [START_REF] Sunahori | The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis[END_REF][START_REF] Sellam | The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis[END_REF] .

Moreover, synovitis is significantly associated with OA severity [START_REF] Loeser | Osteoarthritis: A Disease of the Joint as an Organ[END_REF] . Macrophages are the main immune cell type in the healthy synovium and are likely the front-line cells that sense joint damage. These cells also contribute to OA progression in response to alarmins by producing MMPs and cytokines [START_REF] Raghu | CCL2/CCR2, but not CCL5/CCR5, mediates monocyte recruitment, inflammation and cartilage destruction in osteoarthritis[END_REF] . The main morphological characteristic of synovitis is macrophage accumulation in the intimal lining [START_REF] Bondeson | The role of synovial macrophages and macrophage-produced mediators in driving inflammatory and destructive responses in osteoarthritis[END_REF] .

Macrophages are characterized by heterogeneity and plasticity in response to stimuli from their microenvironment, leading to a continuum of phenotypes where M1/M2 are the 2 extremes [START_REF] Daghestani | Soluble Macrophage Biomarkers Indicate Inflammatory Phenotypes in Patients With Knee Osteoarthritis[END_REF] . M1 macrophages are activated by interferon-γ and lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α), resulting in the secretion of large amounts of proinflammatory cytokines and mediators such as TNFα, interleukin (IL)-1 and IL-6 [START_REF] Sica | Macrophage plasticity and polarization: in vivo veritas[END_REF] . M2 macrophages have been further divided into specific subtypes and possess anti-inflammatory activity [START_REF] Lopa | Arthritic and non-arthritic synovial fluids modulate IL10 and IL1RA gene expression in differentially activated primary human monocytes[END_REF] . Studies have shown that macrophages accumulate and become polarized (M1 or M2) in the synovium during OA development [START_REF] Shapouri-Moghaddam | Macrophage plasticity, polarization, and function in health and disease[END_REF] . The classification of macrophages into M1/M2 subtype is reductive. Recently, the heterogeneity of macrophage phenotypes in OA patients has been studied and revealed a more complex classification [START_REF] Wood | Macrophage proliferation distinguishes 2 subgroups of knee osteoarthritis patients[END_REF] . However, the role of macrophages, their polarization in OA development and the underlying mechanisms are still unknown.

Activated synovial cells (synoviocytes or macrophages) secrete several degenerative enzymes and inflammatory mediators, as well as alarmins [START_REF] Wang | S100A12 levels in synovial fluid may reflect clinical severity in patients with primary knee osteoarthritis[END_REF][START_REF] Manferdini | Adipose stromal cells mediated switching of the pro-inflammatory profile of M1-like macrophages is facilitated by PGE2: in vitro evaluation[END_REF] . These alarmins activate pattern-recognition receptors (PRRs) including TLRs (toll-like receptor) in the OA-affected cartilage and synovium [START_REF] Van Lent | Active involvement of alarmins S100A8 and S100A9 in the regulation of synovial activation and joint destruction during mouse and human osteoarthritis[END_REF][START_REF] Liu-Bryan | The Growing Array of Innate Inflammatory Ignition Switches in Osteoarthritis[END_REF] , which in turn amplifies inflammation and degeneration of cartilage. TLRs are transmembrane receptors that display binding affinity for a variety of DAMPs. According to their ligands, location and signaling pathways, ten different genes can be distinguished in humans [START_REF] Liu-Bryan | Synovium and the Innate Inflammatory Network in Osteoarthritis Progression[END_REF] . TLRs activate signaling pathways that result in the production of cytokines, chemokines, and various inducible molecules associated with the immune response. TLR2 and TLR4 are overexpressed in OA cartilage, and their presence correlates with histopathological damage [START_REF] Bobacz | Toll-like receptors and chondrocytes: The lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of toll-like receptor 4 and antagonized by bone morphogenetic protein 7[END_REF][START_REF] Kim | The catabolic pathway mediated by Toll-like receptors in human osteoarthritic chondrocytes[END_REF] .

Our team identified 14-3-3ε as a novel soluble mediator that is critical in the communication between subchondral bone and cartilage in OA [START_REF] Priam | Identification of Soluble 14-3-3∊ as a Novel Subchondral Bone Mediator Involved in Cartilage Degradation in Osteoarthritis: Soluble 14-3-3∊ in Bone-Cartilage Communication[END_REF] . The 14-3-3 proteins, a family of seven isoforms (β, ε, γ, η, θ, σ, ξ), are involved in a wide range of vital regulatory processes by binding to more than 200 intracellular proteins [START_REF] Sun | 14-3-3 and its binding partners are regulators of protein-protein interactions during spermatogenesis[END_REF] .

Under normal conditions, these proteins reside intracellularly; however, some reports indicate its presence extracellularly. Externalization of 14-3-3 appears to be mediated by a non-classical pathway, similar to IL1-β, due to the absence of a signal peptide sequence [START_REF] Nickel | The mystery of nonclassical protein secretion[END_REF] . 14-3-3 proteins are released into the extracellular space through an exosomal pathway [START_REF] Chavez-Muñoz | Primary human keratinocytes externalize stratifin protein via exosomes[END_REF] . Extracellular 14-3-3s are now thought to play an important role in the pathogenesis of certain inflammatory conditions. In OA, our team showed that 14-3-3ε, released by osteoblasts in response to mechanical stress, skews chondrocytes toward a pro-catabolic phenotype by strongly inducing the expression of MMPs in an CD13/APN-dependent manner [START_REF] Priam | Identification of Soluble 14-3-3∊ as a Novel Subchondral Bone Mediator Involved in Cartilage Degradation in Osteoarthritis: Soluble 14-3-3∊ in Bone-Cartilage Communication[END_REF][START_REF] Nefla | The pro-inflammatory cytokine 14-3-3 is a ligand of CD13 in cartilage[END_REF] . 14-3-3ε seems to share common characteristics with other alarmins (such as ATP and thymosin β4) in inducing degradation of the cartilage matrix [START_REF] Oppenheim | Alarmins: chemotactic activators of immune responses[END_REF] . Extracellular 14-3-3ε proteins could, therefore, be classified as alarmins that are derived from activated or damaged osteoblasts.

Our aim is to investigate the effects of 14-3-3ε on the different cell types of the joint and its role in the activation of innate immunity by studying interactions with its potential receptors TLR2 and TLR4.

Methods

Materials

All reagents were purchased from Sigma-Aldrich (Lyon, France), unless stated otherwise. Fetal bovine serum (FBS) was obtained from Invitrogen (Cergy-Pontoise, France). Liberase TM and complete protease inhibitor mixture were from Roche Diagnostics (Meylan, France). Recombinant human 14-3-3ε was from Enzo Life Sciences. Anti-TLR2 antibody and OXPAPc (oxidized 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphocholine) were from InvivoGen (Toulouse, France). Anti-TLR4

antibody was from Santa Cruz Technology (Heidelberg, Germany.

Collection of human OA synovium

Human OA knee explants were obtained from patients undergoing total knee arthroplasty due to OA at Saint-Antoine Hospital (Paris) or at the Maussins clinic

Cell cultures

Detailed protocols are described in supplemental data and (36).

Synovial explants : For explants, synovium from OA patients was cut into small pieces aseptically and incubate in 24-well plate before treatment.

Primary culture of synovial fibroblasts: Primary cultures of fibroblast like synoviocytes are obtained after enzymatic digestion (collagenase and DNase) of human synovial membrane samples (Biojoint biobank). The digestion solution was then placed in culture flasks during an overnight incubation. The digestion solution was removed, and adherent cells were washed 3 times with phosphate buffered saline (PBS) and cultured in FLS growth medium at 37°C in a humidified atmosphere (5% CO2). After reaching their confluence, FLS were counted and cultured in 12-well plates for stimulations [START_REF] Eymard | Induction of an inflammatory and prodegradative phenotype in autologous fibroblast-like synoviocytes by the infrapatellar fat pad from patients with knee osteoarthritis[END_REF] .

Primary culture of murine articular chondrocytes: Mouse primary chondrocytes were isolated from the articular cartilage of 5 to 6-day-old C57Bl6 mice from Janvier (St.

Berthevin, France). All experiments were performed according to protocols approved by the French and European ethics committees (Comité Régional d'Ethique en Expérimentation Animale N°3 de la région Ile de France). Each littermate among the mice was used for one experiment. After 1 week, the cells were incubated in fasting medium for 24h before treatment.

In addition, experiments were also performed using TLR2-/and TLR4-/mice.

Primary culture of murine bone marrow mononuclear (BMM) cells: Bone marrow mononuclear (BMM) phagocytic precursor cells were isolated from femurs and tibiae of WT and TLR2-/and TLR4-/mice. These precursors were differentiated into adherent mature macrophages (BMM) for 7 days in complete medium containing 10 ng/mL of macrophage colony stimulating factor (PeproTech, Neuilly-sur-Seine, France).

Culture of the THP-1 cell line: Human monocytic THP1 cells (American Type Culture Collection, Rockville, MD, USA) were kind gifts from Professor Rouis (Sorbonne University, France). Cells were routinely grown to a primary macrophage culture following differentiation from monocytes with a 24h treatment with Phorbol 12-Myristate 13-Acetate (50 nM) PMA, Sigma, Saint-Louis, USA). After 72h, the macrophages were starved before treatment.

Treatment by recombinant 14-3-3ε: All cell cultures were stimulated with recombinant 14-3-3ε at 1 µg/ml for 24h. Supernatants and total mRNA collected after cell lysis were harvested and stored at -80°C.

For blocking antibody and pharmacological experiments, murine articular chondrocytes were pretreated for 20 minutes with increasing concentrations (1 and 5 µg/ml) of a mouse TLR2 or TLR4 antibody or oxidized 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphocholine (OxPAPC) at 0.3, 3 and 30 µg/ml before the treatment by recombinant 14-3-3ε.

RNA extraction and quantitative RT-PCR

Total RNA was extracted from murine chondrocytes using the ReliaPrep RNA Cell

Miniprep System kit (Promega, Madison, WI, USA) from human synovial fibroblasts, BMM and THP1 by Trizol chloroform. Concentrations were determined by spectrophotometry (Eppendorf, Le Pecq, France). Reverse transcription was performed with 500 ng of total RNA with the Omniscript RT kit (Qiagen). mRNA levels were quantified with the Light Cycler LC480 (Roche Diagnostics, Indianapolis, IN, USA). PCR amplification conditions are described in supplemental data. Product formation was detected at 72°C in the fluorescein isothiocyanate channel. The mRNA levels were normalized to those of murine HPRT or Human 18S. Specific primer sequences are presented in Table S1.

Protein secretion quantification by ELISA

Total mouse and human IL6, MCP1, TNFα, MMP-3 and 14-3-3ε secretion were assayed in cell-free supernatants using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems and Abbexa) according to the manufacturer's instructions.

Concentrations were analyzed in duplicate at serial dilutions and determined by comparison against a standard curve.

Endotoxin tests:

Protocol of the experiments is described in supplemental data

Statistical Analysis

The choice of the number of experiments was established by power analysis tests and previous and published work from our laboratory. The small number of experiments used for each part of the work can be considered as a limitation. All data were showed as mean values +/-s.e.m. In Fig. 5a and b, the stimulated condition (14-3-3ε) was normalized to 1 in order to study the inhibition rates and data were showed as mean values with 95% confidence intervals (CI). Statistical analyses were performed with the Mann Whitney test to compare mean values between 2 groups or by the Wilcoxon test when analyses were based on patient paired-matched samples (Fig. 1 and2). One-way analysis of variance (ANOVA) and two-way ANOVA with the Bonferroni multiple comparisons post-test were used to compare mean values between more than 2 groups using GraphPad Prism software (GraphPad Software, San Diego, CA). P < 0.05 was considered statistically significant. *P<0.05; **P<0.01; ***P<0.001; ns: not significant.

Results

Stimulation of synovium explants from OA patients by 14-3-3ε elicits the release of pro-inflammatory factors.

To verify whether 14-3-3ε is able to induce synovium inflammation in humans, we used synovium explants from OA patients to mimic the pathophysiological environment as closely as possible. Stimulation of these human synovium explants with recombinant 14-3-3ε (1 µg/ml) induced the secretion of MCP1 and IL6 protein (Fig. 1A,B). Indeed, mean difference of IL6 protein release showed a 5.7-fold increase between control and 14-3-3ε stimulation (0.6, 95%CI [0.3;0.9] vs 3.4, 95%CI [2.7;4.1] respectively) (Fig. 1A) and mean difference of MCP1 protein release showed a 9.5-fold increase between control and 14-3-3ε stimulation (4.8, 95%CI [0.9;8.6] vs 45.6, 95%CI [9.9;81.3] respectively) (Fig. 1B).

14-3-3ε elicits a pro-inflammatory phenotype in fibroblast-like synoviocytes (FLS).

To more precisely study the impact of 14-3-3ε on the two main cell types residing in the synovium, FLS and macrophages, we stimulated primary cultures of FLS from OA patients with 14-3-3ε recombinant protein (1 µg/ml) for 24h. The levels of IL-6 and MCP1 in controls samples are below the detection threshold. We found that stimulated synoviocytes had increased mRNA expression and secretion of both IL6 and MCP1 (Fig. 2 A-D). Mean difference of IL6 mRNA expression showed an increase of 7.5 fold between control and 14-3-3ε stimulation (0. control and 14-3-3ε stimulation respectively (Fig. 2C,D).

14-3-3ε skews macrophages toward a pro-inflammatory phenotype involving

Toll-like receptors signaling.

Stimulation of human macrophages derived from the THP1 cell line with 14-3-3ε induced mRNA expression and secretion of both IL6 and MCP1 (Fig. 3A,B,D,E).

Mean difference of mRNA expression levels of IL6 and MCP1 showed a fold increase of 7.2 (0. control and 14-3-3ε stimulation respectively (Fig. 3D,E,F). Moreover, macrophages subjected to 14-3-3ε stimulation displayed increased mRNA expression of CD38, another marker associated with the M1 pro-inflammatory phenotype, with a 6.6-fold mean difference between control and 14-3-3ε stimulation (0.1, 95%CI [0;0.2] vs 0.9,

95%CI [0.7;1.1]) (Fig. 3C),
To assess the implications of TLR2 and TLR4 in the cellular response to 14-3-3ε, we used primary cultures of bone marrow macrophages (BMM) from TLR2 or TLR4 KO mice and stimulated them with recombinant 14-3-3ε. Untreated WT, TLR2 KO and TLR4 KO BMM showed no protein release of IL6 and MCP1, whereas IL-6 level and MCP1 release were increased by WT BMM cells stimulated with 14-3-3ε. IL-6 protein release was significantly decreased in the supernatants of TLR2 and TLR4 KO BMM stimulated with 14-3-3ε compared to the WT BMM stimulated with 14-3-3ε (64% inhibition for TLR2, WT 2.5, 95%CI [0.9;4.1] vs TLR2 KO 0.9, 95%CI [0.4;1.3] and 84% inhibition for TLR4; TLR4 KO 0.4, 95%CI [0.2;0.6]) (Fig. 4A). MCP1 showed the same tendency (22% inhibition for TLR2 WT 2.8, 95%CI [-2.0;7.7] vs TLR2 KO 2.2, 95%CI [-1.5;6.0] and 68% inhibition for TLR4; TLR4 KO 0.9, 95%CI [-2.1;3.8]) (Fig. 4B).

14-3-3ε elicits a catabolic and inflammatory phenotype in murine articular

chondrocytes involving TLR2 and TLR4.

Murine chondrocytes were sensitive to stimulation with 14-3-3ε recombinant protein and showed increased mRNA expression and secretion of pro-catabolic (MMP3) and pro-inflammatory (IL6) factors.

To confirm the involvement of TLR2 and TLR4 in 14-3-3ε signaling, murine articular chondrocytes were pre-treated with specific TLR2, TLR4 blocking antibody or the pharmacologic inhibitor (OxPAPC) inhibiting both receptors followed by 14-3-3ε stimulation. MMP3 mRNA expression and protein secretion induced by 14-3-3ε were significantly and dose-dependently reduced by TLR4 blocking antibody treatment (MMP3 mRNA expression inhibition fold: 78%, (95%CI [-0.1;0.6]) (1 µg/ml) and 77%

(95%CI [-0.02;0.5]) (5 µg/ml) (Fig. 5A); secretion: 69%, (95%CI [-0.18;0.81]) (1 µg/ml) and 84%, (95%CI [-0.09;0.42]) (5 µg/ml) (Fig. 5D)). Anti-TLR2 blocking antibody reproduced the same pattern (mRNA expression: 79%, (95%CI [-0.01;0.43]) (1 µg/ml) and 83%, (95%CI [0.04;0.30]) (5 µg/ml) (Fig. 5B); secretion 73%, (95%CI [-0.07;0.61]) (1 µg/ml) and 74%, (95%CI [0.04;0.48]) (5 µg/ml) (Fig. 5E)). Furthermore, the pharmacologic inhibitor OxPAPC, inhibiting both receptors, markedly decreased MMP3 mRNA expression and protein release in a dose-dependent manner (mRNA expression: from 71% (95%CI [0.04;0.54]) (0.3 µg/ml) to 98% (95%CI [-0.01;0.04]) (30 µg/ml) (Fig. 5C); secretion: from 64% (95%CI [0.14;0.58]) (0.3 µg/ml) to 96%

(95%CI [-0.01;0.08]) (30 µg/ml) (Fig. 5F)).

Inhibition of TLR2 and/or TLR4 also impacted the mRNA and protein expression of the pro-inflammatory cytokine IL6 by murine chondrocytes. (IL6 mRNA expression:

anti-TLR4 treatment: 73% (95%CI [-0.15;0.68]) (1 µg/ml) and 71% (95%CI [-0.10;0.69]) (5 µg/ml) (Fig. 5G); secretion: 79% (95%CI [-0.12;0.55]) (1 µg/ml) and 81% (95%CI [-0.10;0.48]) (5 µg/ml) (Fig. 5J); anti-TLR2 treatment: IL6 mRNA expression 53% (95%CI [-0.02;0.97]) (1 µg/ml) and 51% (95%CI [0.04;0.94]) (5 µg/ml) (Fig. 5H); secretion 69% (95%CI [-0.03;0.64]) (1 µg/ml) and 78% (95%CI [0.01;0.43]) (5 µg/ml) (Fig. 5K)). Inhibiting both receptors simultaneously with OxPAPC resulted in decreases IL6 expression and secretion (IL6 mRNA expression: from 69% (95%CI [0.01;0.60]) (0.3 µg/ml) to 92% (95%CI [0;0.15]) (30 µg/ml) (Fig. 5I); secretion: from 69% (95%CI [0.05;0.58]) (0.3 µg/ml) to 96% (95%CI [-0.02;0.09]) (30 µg/ml) (Fig. 5L). Stimulations of chondrocytes with the different treatments showed high variability between cultures.

To confirm the results obtained in KO BMM, we also stimulated TLR2 or TLR4 KO murine articular chondrocytes with 14-3-3ε. TLR2 and TLR4 KO murine chondrocytes exhibited decreased mRNA expression and protein release of IL6 but also mRNA expression of MMP3 and MMP13 compared to WT chondrocytes (Fig. 6). IL6 mRNA expression was significantly decreased in TLR2 and TLR4 KO chondrocytes treated with 14-3-3ε compared to the WT chondrocytes (75% for TLR2; WT 65, 95%CI

[-53.7;183.8] vs TLR2 KO 16.5, 95%CI [3.6;29.4] and 97% for TLR4; TLR4 KO 1.8, 95%CI [1.0;2.5]) (Fig. 6B), and its release was greatly attenuated in TLR2 (77% for TLR2 (WT 21.8, 95%CI [-1.5;45.1] vs TLR2 KO 5.0, 95%CI [2.0;8.0]) and TLR4 (by 97% for TLR4, TLR4 KO 0.7, 95%CI [0.5;0.9]) KO chondrocytes in response to 14-3-3ε stimulation respectively (Fig. 6A). Similarly, MMP3 and MMP13 mRNA expression levels were also decreased in TLR2 and TLR4 KO chondrocytes (For MMP3: 60% for 6C and6D). Taken together, these results demonstrate the involvement of both TLR2 and TLR4 receptors in 14-3-3ε signaling.

Discussion

We have recently identified 14-3-3ε as a new soluble mediator involved in deleterious biochemical interactions between bone and cartilage [START_REF] Kim | The catabolic pathway mediated by Toll-like receptors in human osteoarthritic chondrocytes[END_REF] .

In the present study, we showed that 14-3-3ε is a new alarmin and can act particularly in the synovium during OA pathogenesis. Its role in the activation of innate immunity leading to synovitis could be due to interactions with its potential receptors, TLR2 and TLR4.

It is now established that up to 50% of OA patients have synovitis, as demonstrated by magnetic resonance imaging, ultrasonography and arthroscopy [START_REF] Bijlsma | Osteoarthritis: an update with relevance for clinical practice[END_REF] .

Based on these results and to study the involvement of 14-3-3ε in the synovium of the OA joint, we analyzed the effects of 14-3-3ε on whole synovial tissue explants from OA patients by measuring the release of pro-inflammatory factors. We found that IL6 and MCP1 release in culture media was significantly increased after 14-3-3ε stimulation compared with the control explants. In these complete synovial explants from OA patients, the pathophysiological environment was conserved. There was obvious heterogeneity in the degree of inflammation of the synovium between patient samples, which could be due to the different pathological mechanisms leading to OA among patients [START_REF] Van Spil | Osteoarthritis phenotypes and novel therapeutic targets[END_REF][START_REF] Blom | Crucial role of macrophages in matrix metalloproteinase-mediated cartilage destruction during experimental osteoarthritis : Involvement of matrix metalloproteinase 3[END_REF] . Although fewer synovial macrophages are present in OA compared with RA, they are crucial for the production of proinflammatory cytokines such as IL6 [START_REF] Raghu | CCL2/CCR2, but not CCL5/CCR5, mediates monocyte recruitment, inflammation and cartilage destruction in osteoarthritis[END_REF] . Previous studies have shown that selective depletion of synovial macrophages during experimental OA largely reduces cartilage damage and osteophyte formation, which are 2 major hallmarks of OA [START_REF] Ambarus | Systematic validation of specific phenotypic markers for in vitro polarized human macrophages[END_REF] . Thus, we would like to separately analyze the role in synovitis of 2 main cell types residing in the synovium: fibroblast-like synoviocytes (FLS) and macrophages. We found that 14-3-3ε was able to increase the mRNA expression and protein secretion of IL6 (7.5 and 4.3-fold increase respectively) and MCP1 (4.4 and 32-fold increase) in FLS. These cells were able to respond to 14-3-3ε resulting in an inflammatory phenotype. Moreover, Thp1 cells, a human monocyte cell line, were used to address whether 14-3-3ε could polarize these cells toward a pro-inflammatory macrophage phenotype. In our current study, we showed that 14-3-3ε increased the mRNA expression and protein secretion of IL6 (7.2 and 17.8-fold increase respectively) and MCP1 (3.1 and 19-fold increase).

In addition to the experiments on Thp1, we also performed stimulations of primary cultures of murine macrophages (Bone marrow macrophages: BMM) by 14-3-3ε and studied the expression of different markers. mRNA expressions of pro-inflammatory mediators such as iNOS, IL-1β and TNFα were increased whereas mRNA expression of anti-inflammatory marker (EGR2) was decreased in BMM (Fig. S1 Supplemental data). This protein subsequently appears to polarize macrophage toward the M1 phenotype. In the case of knee OA, a study analyzing M1 macrophages and M2 macrophages in synovial fluid in normal versus OA knees found a higher ratio of M1/M2 in OA versus normal knees, and the ratio was significantly correlated to the Kellgren-Lawrence grade [START_REF] Liu | Imbalance of M1/M2 macrophages is linked to severity level of knee osteoarthritis[END_REF] . These results suggest that macrophage polarization may indeed play a role in the control and even progression of OA disease. However, it should be noted that the classification of macrophages into M1/M2 subtype is reductive. This ability of macrophages to modify their phenotype in response to external signals gives rise to a broad spectrum of possibilities depending on their interactions [START_REF] Wood | Macrophage proliferation distinguishes 2 subgroups of knee osteoarthritis patients[END_REF] . Our study indicates that 14-3-3ε can induce a proinflammatory environment in the OA synovium through stimulation of macrophages which possibly contributes to the joint destruction that occurs during OA. Similar results have been obtained previously for the alarmin S100A9 in the synovium [START_REF] Bosch Mh Van Den | Alarmin S100A9 Induces Proinflammatory and Catabolic Effects Predominantly in the M1 Macrophages of Human Osteoarthritic Synovium[END_REF] .

Interestingly, high levels of many alarmins have been described in the synovial fluid of OA patients [START_REF] Tamaki | Expression of Toll-like Receptors and Their Signaling Pathways in Rheumatoid Synovitis[END_REF] . Numerous studies have shown that these DAMPs stimulate synovial cell proliferation, influence hypertrophic chondrocyte differentiation and induce inflammatory and pro-catabolic events in vitro, and they promote synovitis and cartilage degradation in vivo in murine OA models [START_REF] Schelbergen | Alarmins S100A8 and S100A9 elicit a catabolic effect in human osteoarthritic chondrocytes that is dependent on Toll-like receptor 4[END_REF] . We hypothesized that 14-3-3ε is a new alarmin involved in OA. Our results showed that this protein was able to lead to an inflammatory and catabolic response in joint similarly to other alarmins.

Moreover, OA is mainly linked to activation of innate immunity by the binding of damage-associated molecular patterns (DAMPs) to so-called pattern recognition receptors (PRRs) [START_REF] Kawai | The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors[END_REF] . Of central importance in the PRR family are the Toll-like receptors (TLRs) [START_REF] Kawai | The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors[END_REF] . TLR2 and TLR4 are overexpressed in OA cartilage, and their presence correlates with histopathological damage [START_REF] Bobacz | Toll-like receptors and chondrocytes: The lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of toll-like receptor 4 and antagonized by bone morphogenetic protein 7[END_REF] . Blockade of TLR signaling -as shown in TLR2/TLR4 and in MyD88 knockout mice-downregulates cartilage catabolic response in vitro, and it can protect animals from experimental OA [START_REF] Nasi | Dispensable role of myeloid differentiation primary response gene 88 (MyD88) and MyD88-dependent toll-like receptors (TLRs) in a murine model of osteoarthritis[END_REF] .

Moreover, synovial fluid proteins from patients with OA activate macrophages via TLR2/TLR4 receptors, in turn translocating NF-κB to the nucleus 9 . Synovial fibroblasts are sensitive to both mechanical alterations and DAMPs due to the expression of different TLRs on the cell membrane, resulting in increased synthesis of pro-inflammatory mediators [START_REF] Liu-Bryan | The Growing Array of Innate Inflammatory Ignition Switches in Osteoarthritis[END_REF] . In particular, TLR-2 and TLR-4 are used by many alarmins [START_REF] Park | Involvement of Toll-like Receptors 2 and 4 in Cellular Activation by High Mobility Group Box 1 Protein[END_REF] .

To examine whether 14-3-3ε response was driven by TLR, we used TLR2 and TLR4

KO mice to study their potential involvement in macrophage and chondrocyte responses. Our results showed that these two receptors were involved in the inflammatory and catabolic phenotype after stimulation with 14-3-3ε, with TLR4

showing predominant involvement. To further investigate the involvement of TLR2 and TLR4, we used blocking antibodies against them and a pharmacological inhibitor (OXPAPC) that is able to inhibit both TLR2 and TLR4. Our results validated the results obtained in the KO mice and confirmed that 14-3-3ε could elicit a catabolic and inflammatory phenotype in murine articular chondrocytes and a pro-inflammatory phenotype in BMM macrophages. Interestingly, a TLR4 monoclonal antibody has recently been demonstrated to have an adequate safety profile, and a phase II clinical trial in patients with RA has been launched [START_REF] Monnet | Evidence of NI-0101 pharmacological activity, an anti-TLR4 antibody, in a randomized phase I dose escalation study in healthy volunteers receiving LPS[END_REF] .

In the present study, we used recombinant 14-3-3ε protein produced in Escherichia coli, similarly to many commercially available recombinant proteins.

Although this expression system has many advantages, including rapid expression, high yields, ease of culture and low cost [START_REF] Demain | Production of recombinant proteins by microbes and higher organisms[END_REF] , the proteins recovered may be contaminated with endotoxin, a highly complex lipopolysaccharide (LPS) constitutive of the outer membrane of most gram-negative bacteria [START_REF] Heumann | Initial responses to endotoxins and Gram-negative bacteria[END_REF] . LPS is recognized by a receptor complex composed of TLR4, CD14 and MD-2 [START_REF] Triantafilou | Lipopolysaccharide recognition: CD14, TLRs and the LPSactivation cluster[END_REF][START_REF] Pålsson-Mcdermott | Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4[END_REF] . Consequently, using recombinant 14-3-3ε in this study, we wanted to be sure that the effects of 14-3-3ε on joint tissues were due to the protein itself and not to endotoxin contamination. Low levels of endotoxins measured by a LAL kit (14-3-3ε contained less than 0.15 ng/ml of LPS, data not shown) and no significant inhibition by the PMB on 14-3-3ε chondrocyte stimulation (Fig. S2 supplemental data) and 14-3-3ε macrophages stimulation (data not shown) confirmed the proper effect of 14-3-3ε recombinant protein. Moreover, in our previous study [START_REF] Priam | Identification of Soluble 14-3-3∊ as a Novel Subchondral Bone Mediator Involved in Cartilage Degradation in Osteoarthritis: Soluble 14-3-3∊ in Bone-Cartilage Communication[END_REF] , we demonstrated that immunodepletion and blocking of 14-3-3ε in conditioned media of compressed osteoblasts inhibited its catabolic effect on chondrocytes confirming that 14-3-3ε itself is involved in the establishment of a procatabolic phenotype in chondrocytes.

Thus, although we cannot rule out that a small component of the effects observed herein were due to endotoxin contamination, we are confident that the cellular responses resulted from the activity of 14-3-3ε protein itself.

Taken together, our results designate 14-3-3ε as a novel alarmin for further exploration in OA for either therapeutic or prognostic purposes. Stimulation of murine chondrocytes was performed using control medium or 14-3-3ε

Figure legends

(1 µg/ml) treatment. Total RNA was extracted, and mRNA levels of IL6 (B) (n=4), were totally inhibited by PMB. This result show that recombinant 14-3-3ε has its self-1 effect independent from endotoxins. Murine articular chondrocytes cultures were stimulated with 14-3-3ε (1 µg/ml) or LPS (10 ng/ml) with or without PMB (30 µg/ml). Total RNA was extracted and mRNA levels

Fig. 1 :Fig. 2 :

 12 Fig. 1: Stimulation of MCP1 and IL6 protein release by synovium explants from

Fig. 3 :

 3 Fig. 3: Stimulation of IL6, MCP1 and CD38 mRNA expression and IL6, MCP1,

Fig. 4 :Fig. 5a :

 45a Fig. 4: Stimulation of IL6 and MCP1 protein release by bone marrow

Fig 5b :

 5b Fig 5b: Involvement of TLR2 and TLR4 in 14-3-3ε-induced IL-6 (pro-

Fig. 6 :

 6 Fig. 6: Stimulation of IL6 mRNA expression and Il6, MMP3 and MMP13 protein

  MMP3 (C) (n=4) and MMP13 (D) (n=4) were determined by qRT-PCR. Protein levels of IL6 (A) (n=4) in cell supernatants was measured by ELISA. Bars show the mean ± s.e.m. (Two way ANOVA post test)*P<0.05; **P<0.01; ***P<0.001; ns, not significant.

  Figure 1

Figure 6 Relative 2 Fig. S1 :Fig. S2: Assessment of 14 - 3 -

 62S1143 Figure 6

  of MMP13 was determined by qRT-PCR (A) n=3. Protein level of MCP1 in cell supernatants was measured by ELISA (B) n=3. 1% for MCP1 release and 15% for MMP13 mRNA expression, as compared to 100% inhibition between LPS and LPS+PMB in the cellular response, but the major effect of 14-3-3ε was retained even in the presence of polymyxin. (One-way ANOVA with a Bonferroni post test) *P<0.05; **P<0.01; ***P<0.001; ns : not significant.

Table S2 : RT-qPCR amplification conditions

 S2

	Species 95°C: 5min	Name		Forward primer (5'3')	Reverse primer (5'3') Denaturation (1 cycle)
	MMP-3 Denaturation: 10sec at 95°C TGAAAATGAAGGGTCTTCCGG	GCAGAAGCTCCATACCAGCA
	MMP-13 Hybridation of primers: 15sec at 60°C GATGGCACTGCTGACATCAT	TGTAGCCTTTGGAACTGCTT Amplification
	IL6 Elongation: 10sec at 72°C	GTCACAGAAGGAGTGGCTA	AGAGAACAACATAAGTCAGATACC
	MCP-1 95°C during 10sec then 60°C during 30sec CATCCACGTGTTGGCTCA	GATCATCTTGCTGGTGAATGAGT Fusion (1 cycle)
	Murine 40°C: 30sec	CD68 HPRT	AAGATGTTCACCCTGGAGGA AGGACCTCTCGAAGTGT	ACTCCAATGTGGGCAAGAGA ATTCAAATCCCTGAAGTACTCAT Cooling (1 cycle)
		iNOS	AAGGTGAGCTGAACGAGGAG	GGAGCCTTTAGACCTCAACAGA
		IL-1β	GGGCCTCAAAGGAAAGAATC	TCCACTTTGCTCTTGACTTCTATC
		TNFα	CAAGGACTCAAATGGGCTTTCCG TCTGTGAGGAAGGCTGTGCATTG
		EGR2		CTACCCGGTGGAAGACCTC	AATGTTGATCATGCCATCTCC
		18S		GCAATTATTCCCCATGAACG	GGGACTTAATCAACGCAAGC
	Human	IL6 MCP-1	CAATGAGGAGACTTGCCTGG TCAAACTGAAGCTCGCACTCT	GCACAGCTCTGGCTTGTTCC GTGACTGGGGCATTGATTG
		CD38		CCTCACATGGTGTGGTGAAT	TTGCTGCAGTCCTTTCTCC
	Table S1 : PCR Primers

Acknowledgements

The authors thank the Department of Orthopaedic Surgery and Traumatology of Saint-Antoine Hospital for providing human OA tissues. The authors thank Dr. F. Pène (Institut Cochin, INSERM U1016, CNRS UMR8104, Paris, France ; Université

Paris Descartes, Sorbonne Paris Cité, Paris France) for his kind gift of TLR2 and TLR4 KO mice. The authors thank T. Ledent, L. Dinard, A. Guyomard, T. Coulais, and Q. Pointout (animal housing facility, INSERM, Saint-Antoine Research Center, Sorbonne University, Paris) for their excellent work. Contributions -Conception and design: MM, FB, CJ, FP -Collection and assembly of data: MM, LS, AR, AP, MN, CR -Analysis and interpretation of the data: MM, CJ, FB -Drafting of the article: MM, CJ, FB, -Critical revision of the article for important intellectual content: MM, CJ, FB, FP, GA-L, -Final approval of the article: MM, LS, MN, FP, CR, AR, AP, GA-L, FB, CJ Role of the funding source This work was supported by grants from INSERM, Sorbonne University, French Society of Rheumatology (Société Française de rhumatologie) and foundation Arthritis -Courtin . M.M. was supported by a doctoral fellowship from Ministère de l'Enseignement Supérieur et de la Recherche.

Conflict of interest

No potential conflicts of interest relevant to this article were reported. All authors disclose any financial and personal relationships with other people or organizations that could potentially and inappropriately influence their work and conclusions. Murine articular chondrocytes cultures were stimulated with 14-3-3ε (1 µg/ml) or LPS (10 ng/ml) with or without PMB (30 µg/ml). Total RNA was extracted and mRNA levels of MMP13 was determined by qRT-PCR (A) (n=3). Protein level of MCP1 in cell supernatants was measured by ELISA (B) (n=3). Bars show the mean ± s.e.m.

(One-way ANOVA with a Bonferroni post test) *P<0.05; **P<0.01; ***P<0.001; ns: not significant. Treatment with PMB inhibited MMP13 mRNA expression (15% compared to 14-3-3ε stimulation), MCP1 release (1% compared to 14-3-3ε stimulation) whereas MMP13 mRNA expression and MCP1 protein release in response to LPS stimulation