Faisal Syafar
email: faisal@mymail.unisa.edu.au

Jing Gao
email: jing.gao@unisa.edu.au

Mobile Collaboration Technology in Engineering Asset Maintenance -What Technology, Organisation and People Approaches are required?

Keywords: Mobile technology, Collaboration, Engineering asset, Framework 1

Engineering asset maintenance consists of coordinated activities and practices for retaining or restoring a piece of equipment, machine, or system to specified operable conditions to achieve its maximum useful life. An integrated high-level maintenance comprising multiple sub-systems requires the collaboration of many stakeholders including multiple systems and departments. Several of specialised technical, operational and administrative systems have been invested by engineering asset organisations to enhancing their asset management and maintenance systems, however there is no common ground among engineering asset organisations about what are collaborative maintenance are required for adoption/implementation. The lack of systematic approach, together with the lack of specific requirements to implement mobile collaborative maintenance requests a comprehensive framework for guiding engineering organisation to implement of new mobile technologies that meet all maintenance collaboration requirements. This research proposes to develop an appropriate mobile collaboration framework based on Delphi and Case Study investigation.

Introduction

An imperative element of business management is having engineering asset management (EAM). It is essential to realize a business organization's mission which is having or operate assets. EAM is concerned with the life cycle of engineering or physical assets. It is also a critical stage of the continuous life cycle of assets, which includes acquisition, design, installation, maintenance and operation, and disposal stages [1]. The purpose of EAM is to maximise the total benefits of an enterprise by effectively using these assets throughout the whole life cycle.

The importance of maintenance function has increased because it plays an important role in retaining and improving system availability and safety, and product quality [START_REF] Tsang | Strategic Dimension of Maintenance Management[END_REF]. Reference [START_REF] Hodkiewicz | Education in Engineering Asset Management Current Trends and Challenges[END_REF] states that engineering assets in industries rely highly on their maintenance division to maintain and ensure assets are delivered properly. This author also revealed that in the last 30 years, the practice of doing maintenance has significantly changed due to developments in equipment design, information and communication technology, cost pressures, customer acceptance of risk and failures [START_REF] Hodkiewicz | Education in Engineering Asset Management Current Trends and Challenges[END_REF] and the existence of multiple stakeholders and departments [START_REF] Snitkin | Collaborative Asset Lifecycle Management Vision and Strategies[END_REF]. Moreover, current working circumstances are more complex and therefore need to be managed by multiple and interlinked activities [START_REF] Camacho | MobileSJ: Managing Multiple Activities in Mobile Collaborative Working Environments[END_REF]. Hence, an integrated high-level maintenance system which contains multiple sub-systems requires the collaboration of multiple stakeholders such as departments or units to improve resources, information sharing and maintenance practices.

Collaborative maintenance is not a technology or a software solution; rather, it is a customized business strategy unique to each situation [START_REF] Laszkiewics | Collaborative Maintenance: a Strategy to Help Manufactures Become Learn, Mean, and Agile[END_REF]. Based on a review of some relevant references [START_REF] Ferrario | Distributing case-base maintenance: the collaborative maintenance approach[END_REF][8] [START_REF] Rein | Collaboration Technology for Organizational Design[END_REF], it is found that many organizations already have a collaborative maintenance system in place. However, with proper collaboration and commitment, that system can be expanded in scope and effectiveness.

To achieve the quality and efficiency of maintenance for engineering assets, this research proposes a framework to guide the adoption and implementation of new mobile technologies. It aims to facilitate asset maintenance collaboration, where organizations can expand their existing technology. In this context, efficiency means maintaining engineering assets without interrupting the production process for unnecessary breakdowns.

This research is structured as follows: the first section describes the motivation of this research. The second section provides a brief review of the concept of mobile collaboration technology for engineering or physical asset maintenance (PAM). This is followed by a brief description of the proposed framework, research methods, and the last section outlines a conclusion.

Research Motivation

This research is motivated by five factors. First, today's asset maintenance practices rely on access to information and team expertise from dispersed sites

Literature Review

Engineering Asset Maintenance

Maintenance is a combination of actions intended to retain an item in, or restore it to, a state in which it can perform the function that is required for the item to provide a given service. This concept leads to a first classification of the maintenance actions in two main types: actions oriented towards retaining certain operating conditions of an item and actions dedicated to restoring the item to supposed conditions. "Retention" and "restoration" are denominations for action types that are then converted into "Preventive" and "Corrective" maintenance types in the maintenance terminology by European Committee for Standardization.

Reference [START_REF] Márquez | The Maintenance Management Framework[END_REF] acknowledges that maintenance processes consists of several tasks that must be in line with three levels of business activities, namely: strategic, tactical and operational. At the strategic level, business priorities will be transformed into maintenance priorities. The transformation process is done by supporting expertise within a certain period to address current and/or potential gaps in equipment maintenance action or strategy. A generic maintenance plans (middle and long range) will be obtained at this level. Actions at the tactical level will define the appropriate task of maintenance resources such as skills, materials, test equipment, etc. to fulfil a business's maintenance plan. At this level, a detailed program consists of particular tasks and the resources allocated would be achieved. At the operational level, maintenance tasks would be done accurately by skilled engineers, in the time that has been allocated, following right procedures, and using the appropriate tools. Preventive and corrective maintenance procedures are carried out so tasks receive a high level of attention. All maintenance tasks will be completed and the maintenance data history would be recorded in the information system at this level.

Current IT/IS Capabilities for Engineering Asset Maintenance

Reference [START_REF] Sokianos | Lexikon Produktions Management[END_REF] emphasise that in order to manage the sophisticated AM process and to provide its data requirements, particular technology and systems are required. The system that captures, maintains, and manages all the needed asset information throughout the entire asset lifecycle is critical in providing effective asset management. Currently, several of specialised technical and operational systems have been invested by EAM organisations to enhancing their asset maintenance systems. These technologies and systems aimed at support the whole asset lifecycle. The very popular maintenance information systems that have been implementing for engineering asset maintenance are Computerised Maintenance Management Systems (CMMS) [START_REF] Tam | Optimisation Framework for Asset Maintenance investment[END_REF]. However, although CMMS makes a great volume of information available for reliability and efficiency analysis of the delivery of the maintenance function, most experts agree that successful CMMS is less than 30% of total CMMS applications [START_REF] Zhang | CMMS and its Application in Power System[END_REF]. The main reasons are [START_REF] Olszwesky | RCM success starts with CMMS[END_REF]: Selection errors , insufficient commitment, lack of training , failure to address organizational implications, underestimating the project task, lack of project resources and lack of demonstrable use of system output

Collaboration Requirements by Asset Maintenance' Stakeholders

Mobile technologies play a key role in this setting, facilitating to establish tightly integrated environments between different groups and organizations that bear stakes on the performance of the industrial assets [START_REF] Liang | Adoption of Mobile Technology in Business: a Fit-Viability Model[END_REF]. Despite the fact that the use of advanced application solutions in manufacturing, production, or process facilities takes place at a different scale, the emerging trend has already shown that mobile technologies have a great potential to redefine and re-engineer the conventional setting. They have already begun to offer advanced and smart solutions to remotely manage complex, highrisk, and capital-intensive assets, regardless of the geographical location, building agile information and knowledge networks [START_REF] Monostori | Agent-Based Systems for Manufacturing[END_REF].

In order to encounter good asset maintenance and meet the optimum performance of engineering assets, organisations require a collaborative teamwork within key functional areas (stakeholders) of the engineering organisations. Shared understanding, coordination, cooperation and collaboration across maintenance stakeholders of what asset maintenance is and how the entire maintenance team influence the ability to achieve organisational objectives through those assets are one of the critical success factors of asset management. Collaborative asset maintenance is applicable to all those who have a role in the maintenance of engineering assets including directors, managers, supervisor, engineers, IT and maintenance technicians.

Mobile collaboration technology required for asset maintenance need to be capable of simultaneously handling, processing and delivering technical and operational information to multiple maintenance crew at multiple locations at any time to enhance asset maintenance planning and implementation within the three levels of business activities. The requirements are including technological, organisational, as well as personal perspectives.

Proposed Framework

In order to develop the mobile collaboration PAM framework, the research questions need to be answered. The major question is: How can mobile collaboration technologies assist asset maintenance in engineering asset management organization? Based on the extensive literature review, a conceptual research framework was developed as shown in Figure 1. It encapsulates the core concept of [START_REF] Linstone | Decision Making for Technology Executives: Using Multiple Perspectives to Improve Performance[END_REF] TOP model as a means of studying collaboration requirements from either technical or organisational or personal perspectives. It also includes the alignment of maintenance processes with three levels of business activities: strategic, tactical and operational [START_REF] Márquez | The Maintenance Management Framework[END_REF].This conceptual framework will guide the planning and activities in the subsequent research for investigating collaboration requirements in physical asset maintenance.

Fig. 1. Preliminary Maintenance Collaboration Framework

Research Methods

This research will be an interpretive study using both quantitative and qualitative methodologies. References [START_REF] Klein | A Set of Principles for Conducting and Evaluating Interpretive Field Studies in Information Systems[END_REF][19] [START_REF] Orlikowski | Studying Information Technology in Organizations: research approaches and assumptions[END_REF] have reasoned that interpretive attempts to understand phenomena through the meanings that people assign to them are relevant. This understanding is particularly relevant in this research because the researcher is seeking to understand certain issues by industries survey, Delphi study and interviewing people on how mobile collaboration technologies will assist the asset maintenance process in a given organization's context. In order to create a complete set of requirements of collaboration maintenance in engineering organizations, the case study results here will be triangulated with the survey, Delphi study and case study findings.

Triangulation is the use of more than one research strategy to explore the same phenomenon so that the credibility of research results is improved [START_REF] Greene | Making Paradigmatic Sense of Mixed Methods Practice[END_REF].

The Delphi Technique

This study is conducted to identify collaboration requirements, current collaborative maintenance practice and mobile technology roles in support collaborative engineering asset maintenance. The Delphi technique is employed to more accurately build the consensus from the panel expert's perception [START_REF] Dalkey | An Experimental Application of the Delphi Method to the Use of Experts[END_REF]. The Delphi study is a group process to solicit expert responses toward reaching consensus on a particular problem, topic, or issue by subjecting them to a series of in-depth questionnaires, interspersed with controlled feedback [START_REF] Dalkey | An Experimental Application of the Delphi Method to the Use of Experts[END_REF]. The Delphi method is employed for several reasons. The topic 'Mobile collaboration technology in engineering asset maintenance' is quite new, it is complex, a few literatures series have been found, and not much empirical data was available. Those are the reasons why Delphi study is useful to confront a mobile maintenance expert's panel. Delphi study is carried out in this research which comprised three rounds [START_REF] Linstone | The DelphiMethod: Techniques and Applications[END_REF]. Nomination of experts. A total of 47 experts who have strong academic backgrounds, research experience and professional in the area of mobile asset maintenance were invited to participate in the Delphi survey. Of these, 20 are willing to participate in the research project. They are 8 from universities and 12 professionals worldwide. The expert's profiles are illustrated below in Table 1 and2 Respondents were asked mainly about three basic questions, each corresponding to one of the research questions. The questionnaire asked experts to list general and the collaborative asset maintenance specific requirements, selecting criteria, benefits as well as initiatives issue that my hinder maintenance collaboration. The second round is validation categorized list of requirements. The experts were asked to verify the list that the researcher have correctly interpreted and placed them in an appropriate category/group based upon first round responses. In this round the experts were also requested to remove, added or regrouped the item (s) into other group/category. The third round is ranking relevant requirements. The consensus in the ranking order of the relevant group/category about requirements will be achieved in this final iteration. They will also be asked about the correlation between requirements (if any) as well as the critical requirements that need to be focus on.

Multiple Case Studies

Semi-structured interview-based multiple case studies will be conducted to explore the collaboration requirements for asset maintenance practices, to obtain information on the deficiencies in existing collaboration requirements.

In order to create a complete set of requirements of collaboration maintenance in engineering organizations, the case study results here will be triangulated with the Delphi study findings. Triangulation is the use of more than one research strategy to explore the same phenomenon so that the credibility of research results is improved [START_REF] Greene | Making Paradigmatic Sense of Mixed Methods Practice[END_REF].

Preliminary Findings and Discussion

Delphi question 1: Please mention collaborative maintenance requirements.

From the responses of 19 panel members, we analyzed 63 individual statements. We then grouped into similar requirements and then mapped into Technology (T), Organization (O) and People (P) approaches as illustrated in Table 3.

Delphi question 2: Please mention the current role of mobile technology in support asset maintenance collaboration technical/system

We coded 19 responses into 42 individual statements. The statements were then clustered by similarity into categories and finally mapped to high-level feature areas as can be seen in Table 4. Visualising of collected data, parameter history and trending. Contextualising access over remote data and services. Critical for response time for data or information that can lead to early correction and or identification of failures. Providing the notification of failure through mobile devices Detecting the location of skilled maintenance personel nearby an asset that has experienced a failure through GPS. Mobile technology allows at the right place to access directly to a set of information coming from all the potential actors involved in the decision (CMMS, ERP, sensors, etc.).

Empowering management

Resources management (material, maintenance people) facilitator for continous task monitoring/assignment/reporting. Building and identifying process verification tasks, approvals.

It helps to report failure effectively and report labors actual working hours and availability. Allowing to take the right maintenance decision, at the right time, at the right place, from the right information. Enhancing accuracy of critical data entry for maintenance history. Off-site (not in office) notifications and live feeds. Q/A decisions Others early adopters stage in the technology livecycle. stage.

Extremely limited use at the moment

Conclusion

Through the development of mobile technologies, the processing of information can be performed by technical personnel away from the central production office or site. Maintenance personnel, when doing their tasks, require relevant information in different sites and need to communicate interactively with experts in the back office. Using mobiles allows maintenance personnel to continuously receive a daily schedule from the head office. This leads to the saving of time and improving customer service and profitability. Furthermore, it is expected that the research finding will develop a unique framework that addresses the following issues (1) Business process alignment at all three levels (strategic, tactical and operational) in company activities through the variable of mobile collaboration technologies, (2) Engineering asset management with a specific focus on the most critical processasset maintenance, and (3) Comprehensive framework that meet all requirements (technological, organisational and people perspectives).

Table 1 .

 1 . Participants by Role

	Background of expert		Participants
		Frequency	Percentage (%)
	Academia	8	40
	Professional	12	60
	Total	20	100

Table 2 .

 2 Participants by Geographic Location

	Location of expert	Participants
		Frequency	Percentage (%)
	Australia	2	10
	Canada	3	15
	France	1	5
	Germany	1	5
	Greece	1	5
	Malawi	1	5
	Qatar	1	5
	Singapore	1	5
	United Arab Emirates	1	5
	US	8	40
	Total	20	100

Delphi Design. Three-round Delphi email-based questionnaire is designed. The first round is initial collection of requirements consisted of open-ended solicitation of ideas.

Table 3 .

 3 Collaborative maintenance Requirements

	TOP	Delphi Round 1
		1. Synchronised multi-user access over a feature-populated
		dashboard
		2. Data and services access through contextualised and
		mobile interfaces
		3. Data and services functionality porting to the cloud
		4. Autonomous information/communication exchange
	Technology	5. Linking the maintenance planning and dispatching
		6. Provides different mode for specific maintenance role
		7. Support interoperability between maintenance role
		8. Provide a platform for maintenance knowledge sharing
		across maintenance crew
		9. Social networking

Table 4 .

 4 Mobile technology roles

	Area	Feature category
	Flexibility
	(initiate
	application
	at flexible
	sites in un-
	structured
	networked)


François Hermetet

Elise Jacquin

Sophie Launay

Emilie Gaiffe

Fabienne Hirchaud

Patrick Sandoz

Jean-Luc Prétet

Christiane Mougin
email: christiane.mougin@univ-fcomte.fr

Efferocytosis of apoptotic HPV positive cervical cancer cells by human primary fibroblasts

Keywords: Human primary fibroblasts, Efferocytosis, Apoptosis, Apoptotic cervical cancer cells, Phosphatidylserine, Brainspecific Angiogenesis Inhibitor-1

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

Introduction:

Clearance of apoptotic cells (AC), so-called efferocytosis, largely contributes to tissue homeostasis in vivo. Although this fundamental physiological process is a property of most cell types [START_REF] Flannagan | The cell biology of phagocytosis[END_REF][START_REF] Underhill | Information processing during phagocytosis[END_REF][START_REF] Savill | Corpse clearance defines the meaning of cell death[END_REF][START_REF] Fadok | Phagocyte receptors for apoptotic cells: recognition, uptake, and consequences[END_REF][START_REF] Erwig | Clearance of apoptotic cells by phagocytes[END_REF][START_REF] Hochreiter-Hufford | Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion[END_REF], macrophages are the main and most effective phagocytes for the removal of AC. They have been widely used to investigate the molecular mechanisms underlying recognition, uptake, and degradation of AC [START_REF] Flannagan | The cell biology of phagocytosis[END_REF][START_REF] Underhill | Information processing during phagocytosis[END_REF][START_REF] Fadok | Phagocyte receptors for apoptotic cells: recognition, uptake, and consequences[END_REF][START_REF] Hochreiter-Hufford | Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion[END_REF][START_REF] Wermeling | Class A scavenger receptors regulate tolerance against apoptotic cells, and autoantibodies against these receptors are predictive of systemic lupus[END_REF][START_REF] Fadok | Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases[END_REF][START_REF] Fadok | Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF[END_REF][START_REF] Voll | Immunosuppressive effects of apoptotic cells[END_REF].

The recognition of AC by macrophages involves numerous receptors expressed on macrophage cell surface that bind their specific ligands exposed on AC and referred to as "Eat me" signals [START_REF] Flannagan | The cell biology of phagocytosis[END_REF][START_REF] Underhill | Information processing during phagocytosis[END_REF][START_REF] Hochreiter-Hufford | Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion[END_REF][START_REF] Lauber | Clearance of apoptotic cells: getting rid of the corpses[END_REF][START_REF] Mevorach | Complement-dependent clearance of apoptotic cells by human macrophages[END_REF]. To date, various "Eat me" signals have been described including altered sugars, lipids and proteins [START_REF] Hochreiter-Hufford | Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion[END_REF] but Phosphatidylserine (PtdSer) is the best-studied [START_REF] Fadok | Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts[END_REF][START_REF] Fadok | Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages[END_REF][START_REF] Hoffmann | Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells[END_REF][START_REF] Krahling | Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages[END_REF][START_REF] Fadok | The role of phosphatidylserine in recognition of apoptotic cells by phagocytes[END_REF]. Previous publications support the importance of PtdSer receptors, including T cell Immunoglobulin-and-mucin domain Molecule (TIM), Brain-specific Angiogenesis Inhibitor 1 (BAI1), and stabilin-2, in the clearance of AC through the direct recognition of externalized PtdSer on the AC surface [START_REF] Fadok | A receptor for phosphatidylserine-specific clearance of apoptotic cells[END_REF][START_REF] Kobayashi | TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells[END_REF][START_REF] Park | The phosphatidylserine receptor TIM-4 does not mediate direct signaling[END_REF][START_REF] Park | BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module[END_REF][START_REF] Park | Stabilin-1 mediates phosphatidylserine-dependent clearance of cell corpses in alternatively activated macrophages[END_REF][START_REF] Park | Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor[END_REF]. TIM-4, highly expressed by macrophage cell lines, is a small transmembrane protein that binds PtdSer via its immunoglobulin domain [START_REF] Kobayashi | TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells[END_REF][START_REF] Miyanishi | Identification of Tim4 as a phosphatidylserine receptor[END_REF]. Bone marrow-derived macrophages from TIM-4 -/- mice were recently reported to have a highly reduced ability to engulf apoptotic bodies or PtdSer-bearing targets [START_REF] Martinez | Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells[END_REF][START_REF] Flannagan | The phosphatidylserine receptor TIM4 utilizes integrins as coreceptors to effect phagocytosis[END_REF]. Such observations suggest that TIM-4 is the primary receptor for PtdSer in those professional phagocytosis models. BAI1, another PtdSer receptor, is a seven transmembrane receptor that belongs to a subfamily of adhesion G-protein-coupled receptors [START_REF] Stephenson | The BAI subfamily of adhesion GPCRs: synaptic regulation and beyond[END_REF]. The first recognized function of BAI1 was its ability to inhibit angiogenesis and tumor formation through the proteolytic processing of its N-terminal domain into secreted vaculostatins [START_REF] Cork | Emerging roles for the BAI1 protein family in the regulation of phagocytosis, synaptogenesis, neurovasculature, and tumor development[END_REF][START_REF] Kaur | Vasculostatin, a proteolytic fragment of brain angiogenesis inhibitor 1, is an antiangiogenic and antitumorigenic factor[END_REF][START_REF] Kaur | Vasculostatin inhibits intracranial glioma growth and negatively regulates in vivo angiogenesis through a CD36-dependent mechanism[END_REF]. BAI1 is expressed in macrophage cell lines and immunological tissues such as the bone marrow and spleen and is known to contribute to AC phagocytosis and myoblast fusion via its thrombospondin (TSP) type 1 repeats [START_REF] Park | BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module[END_REF][START_REF] Hochreiter-Hufford | Phosphatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion[END_REF]. Other receptors [e.g. vitronectin receptor (αvβ3 integrin), Mer tyrosine kinase family (MerTK, Tyro3 and Axl)] are involved in indirect recognition of PtdSer and require soluble bridging proteins [e.g. TSP 1 or Milk Fat Globule-EGF factor 8 (MFG-E8), Gas6 or protein S] [START_REF] Hochreiter-Hufford | Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion[END_REF].

Engagement of the PtdSer receptors initiates signaling events within the phagocyte. The Rho-family GTPases are prominent players in the transmission and integration of signals and activate actin-related protein-2/3 (Arp2/3) complex, formins and WH2 domain-containing nucleators [START_REF] Kuhn | Formins as effector proteins of Rho GTPases[END_REF][START_REF] Pollard | Regulation of actin filament assembly by Arp2/3 complex and formins[END_REF][START_REF] Campellone | A nucleator arms race: cellular control of actin assembly[END_REF]. This leads to actin cytoskeleton remodeling, engulfment of dead cells into macroendocytic vacuole [START_REF] Flannagan | The cell biology of phagocytosis[END_REF][START_REF] Park | BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module[END_REF][START_REF] Swanson | Shaping cups into phagosomes and macropinosomes[END_REF].

Regarding the internalization of dead cells by professional phagocytes, different mechanisms are now being appreciated, even if information about their target cell specificity (apoptotis and necrosis) remains sparse (reviewed in Ref. [START_REF] Krysko | Clearance of apoptotic and necrotic cells and its immunological consequences[END_REF]). Indeed, several observations indicate that the interaction of macrophages with dead cells initiates internalization of the targets, and that internalization can be preceded by phagocytosis "zipper"-like mechanism (i.e. related to a formation of a close-fitting phagocytic cups) or macropinocytotic mechanism (i.e. related to a formation of spacious macropinosomes accompanied by ruffling of the ingesting macrophages).

During macroendocytic maturation, their membrane fuses with intracellular organelles including early endosomes, late endosomes and lysosomes [START_REF] Henry | The uniformity of phagosome maturation in macrophages[END_REF][START_REF] Kinchen | Phagosome maturation: going through the acid test[END_REF][START_REF] Rogers | The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum[END_REF]. Recently, Martinez and colleagues highlighted that efficient clearance of AC by macrophages involves the recruitment and lipidation of Microtubule-associated protein 1 light chain 3 alpha (LC3) to the AC-containing phagosome membrane followed by an acidification of the phagocytic vacuole [START_REF] Martinez | Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells[END_REF]. Authors suggested that during this process named LC3-associated phagocytosis (LAP), LC3 facilitates the phagosome maturation and the degradation of cellular corpses.

AC clearance by non-specialized phagocytic cells has been investigated in a limited number of studies [START_REF] Dini | Phagocytosis of apoptotic bodies by liver endothelial cells[END_REF][START_REF] Hall | Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin[END_REF][START_REF] Monks | Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release[END_REF][START_REF] Patel | Recognition of apoptotic cells by epithelial cells: conserved versus tissue-specific signaling responses[END_REF][START_REF] Wagner | Phagocytosis of dying tumor cells by human peritoneal mesothelial cells[END_REF][START_REF] Viegas | Comparison of the kinetics of maturation of phagosomes containing apoptotic cells and IgG-opsonized particles[END_REF], the results of which suggest that mechanisms underlying prey cell recognition and clearance may differ from those observed in professional phagocytes [START_REF] Flannagan | The cell biology of phagocytosis[END_REF][START_REF] Underhill | Information processing during phagocytosis[END_REF][START_REF] Erwig | Clearance of apoptotic cells by phagocytes[END_REF][START_REF] Lauber | Clearance of apoptotic cells: getting rid of the corpses[END_REF].

Through the endocytic process, AC can act as a DNA vector that may confer a selective advantage to the recipient cell [START_REF] Bergsmedh | Horizontal transfer of oncogenes by uptake of apoptotic bodies[END_REF][START_REF] Ehnfors | Horizontal transfer of tumor DNA to endothelial cells in vivo[END_REF]. In a previous study, we provided evidence of an engulfment of apoptotic human cervical cancer-derived cells by human primary fibroblasts (HPFs) [START_REF] Gaiffe | Apoptotic HPV positive cancer cells exhibit transforming properties[END_REF]. Apoptotic HPV-positive cells ensured a horizontal transfer of oncogenes to HPFs revealed by in situ hybridization and PCR analyses. HPFs subsequently expressed HPV E6 mRNA and exhibited transformed cell properties such as increased proliferation, anchorageindependent growth capacity and aneuploidy. The results highlighted an alternative mechanism of HPVassociated carcinogenesis from mesenchymal cells that are not natural targets of HPV [START_REF] Gaiffe | Apoptotic HPV positive cancer cells exhibit transforming properties[END_REF][START_REF] Grayson | Carcinosarcoma of the uterine cervix: a report of eight cases with immunohistochemical analysis and evaluation of human papillomavirus status[END_REF][START_REF] Unger | An unusual cervical carcinoma showing exception to epitheliotropism of human papillomavirus[END_REF].

In the present study, we have analyzed mechanisms that drive non-professional phagocytes, namely HPFs, to clear apoptotic HPV-positive cervical cancer cells. By using flow cytometry, confocal imaging and functional studies, we show that HPFs are able to phagocytize dead tumor cell corpses by recognizing and binding PtdSer thanks to the specific receptor BAI1 expressed by HFP, engaging actin-fibers remodeling to the macroendocytic vacuole formation and tranlocating LC3 for its maturation.

Results

:

Characterization of apoptotic cell death

The apoptosis of cervical carcinoma donor cells was induced by ultra-violet (UV)-B exposure followed by staurosporine (STS) treatment for 6 h and 48 h as previously described [START_REF] Gaiffe | Apoptotic HPV positive cancer cells exhibit transforming properties[END_REF]. To ensure that only dying and dead cells were used for endocytosis assays, sloughed cells were collected and characterized by apoptosis assays.

By contrast, viable HeLa and Ca Ski used as untreated cell controls were harvested by trypzinisation. As shown by Sub-G1 analysis, the percentage of cells with fragmented DNA increased in a time-dependent manner; no obvious feature of apoptosis or hypodiploid cells was detected in untreated cells (Figure 1A). Apoptosis was further confirmed by specific nuclear morphology changes evidenced by DAPI staining (Figure 1B). Flow cytometry analysis demonstrates that treatment for 6 h resulted in an increase of early apoptotic cells (EAC) defined as annexin V single-positive (74.2% ± 3.8 for HeLa; 80.8% ± 1.0 for Ca Ski) and treatment for 48 h resulted in a high rate of late apoptotic cells (LAC) defined as annexin V and PI double-positive (97.0% ± 1.9 for HeLa; 88.6% ± 3.9 for Ca Ski) (Figure 1C).

Late apoptotic cells are preferentially engulfed by HPFs

To establish the extent to which HPFs could engulf apoptotic cervical carcinoma HeLa and Ca Ski cells regardless of various stages of apoptosis, HPFs were co-cultured with PKH67-labeled EAC or LAC, washed twice to remove adherent cells, fixed in PFA, and labeled with phalloidin-TRITC and DAPI. Based on confocal microscopy analysis, unloaded HPFs could be clearly distinguished from HPFs having ingested early or late PKH67-labeled apoptotic HeLa or Ca Ski cells. Figure 2A is representative of confocal microscopy images of co-culture. It has been established that late apoptotic/necrotic cells induce phagocyte activation, whereas early apoptotic cells do not [START_REF] Savill | Corpse clearance defines the meaning of cell death[END_REF]. As might be expected, the engulfment of EAC analyzed by confocal microscopy was much less efficient than that of LAC (Figure 2A andB). Indeed, although EAC and LAC were co-cultured with HPFs at similar ratio (10:1), most of EAC were released following washing and the remaining cells mostly displayed cell-cell contact but were not phagocytosed as shown in Figure 2A, left panel. The 6 h co-culture duration was chosen for further experiments in order to maintain transient nature of the early apoptotic signature throughout period of co-culture and to avoid appearance of LAC [START_REF] Silva | Secondary necrosis: the natural outcome of the complete apoptotic program[END_REF]. LAC were mostly engulfed by HPFs, as soon as 2 h of co-culture (Figure 2B), as demonstrated by the presence of scattered yellow/green AC within the red HPFs (Figure 2A, middle panel). This result demonstrates that cell fragments (1 to 10 µm in diameter) were unequivocally inside the HPFs. Moreover, the human monocytes-derived macrophages (Mφ), serving as professional phagocyte controls, exhibited their high capacity for the uptake of LAC compared to HPFs (Figure 2A, right panel).

Using the quantitative flow cytometry approach, we assessed the percentage of PKH26-stained HPFs that engulfed PKH67-stained apoptotic cancer cells at early time periods (2 h and 6 h) of co-culture. As shown by the appearance of PKH67-and PKH26 double-positive populations (Figure 2C), the cytometry analyses confirmed the rate of AC uptake by HPFs observed by microscopy (Figure 2C andD). Taken together, these results show that HPFs clearly distinguished EAC and LAC and that their phagocytic capacity was limited to apoptotic subcellular fragments (LAC), which is consistent with their non-professional nature [START_REF] Dini | Phagocytosis of apoptotic bodies by liver endothelial cells[END_REF].

We next studied the kinetics of AC internalization by HPFs and Mφ by varying the exposure time (2 -72 h) of LAC to the phagocytic cells. Again, the phagocytic capacity was defined as the percentage of HPFs and Mφ that engulf LAC. Figure 2E indicates that HPFs and Mφ reached their maximum phagocytic capacity at 24 h post-exposure. Nevertheless, the percentage of phagocytosing HPFs was less compared to Mφ (Figure 2E). In total HPFs engulfed AC with a lower efficiency than Mφ, an observation that further substantiates their amateur phagocyte nature [START_REF] Parnaik | Differences between the clearance of apoptotic cells by professional and non-professional phagocytes[END_REF].

Moreover, we studied in detail morphological aspects of dead cell engulfment by HPFs using confocal microscopy (Figure 2F). The reorganization of the actin cytoskeleton observed during internalization of double-PKH67/DAPI-labeled LAC suggests both phagocytosis and macropinocytotic process. During the initial step of endocytosis, LAC bound to the plasma membrane. Actin polymerization was localized underneath the plasma membrane in contact with LAC to be ingested, and dense network of actin filaments appeared in this region (Figure 2Fa). Then membranous expansions extended around the dead cell (Figure 2Fb) to ultimately engulf it (Figure 2Fd). Macropinocytosis-like movements were also observed (Figure 2Fc). The ingested LAC was completely enclosed into HPFs within 6 h, in a membrane-bound vacuole (Figure 2Fe andf).

In addition, the monitoring of AC clearance was evaluated by position-referenced microscopy as we published [START_REF] Galeano | Position-referenced microscopy for live cell culture monitoring[END_REF]. By tracking the fluorescent dyes during co-culture between PKH26-labeled HPFs and CFSElabeled HeLa LAC at different time periods, we observed that apoptotic corpses quickly adhered (1 h) to the plasma membrane of HPFs and were efficiently internalized by HPFs within 6 h after first tethering (Figure 2G and Figure S1). These time-lapse events have been already described for other non-professional phagocytes [START_REF] Monks | Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release[END_REF][START_REF] Parnaik | Differences between the clearance of apoptotic cells by professional and non-professional phagocytes[END_REF].

Clearance of LAC by HPFs is inhibited by low temperature incubation, EDTA, cytochalasin D and dimethylamiloride

The surface morphology as visualized by confocal microscopy revealed dynamic actin-based membrane protrusions of HPFs that induce phagocytic cup formation during the process of AC internalization. To better understand the type of endocytosis used by HPFs to engulf AC, we tested physical and chemical inhibitors.

When recipient cells were co-incubated with HeLa and Ca Ski AC at 4°C or in the presence of 2 mM EDTA, the percentage of internalization significantly dropped to 2% suggesting that internalization requires energy and divalent ions (Ca 2+ , Mg 2+ ) (Figure 3A). Cytochalasin D (a F-actin depolymerizing agent) or dimethylamiloride (DMA; an inhibitor of Na + /H + exchange that prevents membrane ruffling and macropinocytosis) have been used

to treat HPFs prior to the addition of LAC. These treatments resulted in significant decreases of LAC uptake by HPFs in a dose-dependent manner, suggesting the involvement of phagocytosis and/or macropinocytosis in the internalization process (Figure 3A). A partial inhibition of LAC uptake was seen using chlorpromazine (an inhibitor of clathrin-dependent endocytosis) at a concentration of 20 µM. By contrast nystatin (an inhibitor of caveolin-dependent endocytosis) had no effect on the uptake of LAC whatever the concentration used (Figure 3A). Taken together, these data suggest the main contribution of phagocytosis and macropinocytosis in AC clearance.

It is noteworthy that 6 h treatment with high concentrations of cytochalasin D (> 4 µM), DMA (> 80 µM), chlorpromazine (> 20 µM) and nystatin (> 20 µM) interfered with HPFs viability, as monitored by MTT test (Figure S2), and were not considered for internalization assays.

Efferocytosis of LAC by HPFs is partially mediated by phosphatidylserine and BAI1

Phosphatidylserine (PtdSer) exposure is widely considered as a canonical marker of apoptosis. The contribution of PtdSer recognition by HPFs was evaluated by measuring the ability of HPFs to engulf PKH67-AC incubated with an excess of annexin V. Pretreatment with 40 and 160 µg/mL of annexin V efficiently block PtdSer recognition (Figure 3B) and resulted in a dose-dependent decrease of LAC uptake by HPFs (Figure 3C).

Four main PtdSer receptors have been shown to be involved in the uptake of AC by professional phagocytes:

BAI1, TIM-1, TIM-4 and Stabiline 2 [START_REF] Flannagan | The cell biology of phagocytosis[END_REF][START_REF] Erwig | Clearance of apoptotic cells by phagocytes[END_REF]. Among these 4 different PtdSer receptors, we noted that 2 were expressed by Mφ, namely BAI1 and TIM-4 as shown in RT-qPCR analysis (Figure 3D). The receptors involved in endocytosis of AC by amateur phagocytes have not been clearly identified. Here, HPFs expressed only BAI1 that displayed lower levels of mRNA compared to Mφ (Figure 3D). Moreover, immuno-localization studies with the H270 antibody indicated that BAI1 was not uniformly distributed but showed a variable and patchy membrane staining in HPFs, as well as in Mφ, in absence of apoptotic cells (Figure 3E). During co-culture between HPFs and AC, BAI1 showed a tendency to accumulate within the phagocytic cup (Figure 3F). We raised the hypothesis that fibroblastic BAI1 may subserve an engulfment function of apoptotic corpses. To assess the function of BAI1 in this system we performed short interference RNA (siRNA) transfection in HPFs prior to co-culture as described in detail in material and methods. This resulted in a significant decrease of human BAI1 mRNA levels (Figure 3G). Compared to siRNA negative control, HPFs transfected with BAI1

siRNA demonstrated a lower ability to internalize PKH67-labeled LAC, either HeLa or Ca Ski LAC (Figure 3H). Thus we believe BAI1 to be a surrogate molecule to engulf apoptotic targets.

Recruitment of LC3 onto the phagocytic vacuole occurs after LAC efferocytosis by HPFs

Some components of the autophagy machinery being involved in the maturation of macroendocytic vacuoles [START_REF] Martinez | Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells[END_REF][START_REF] Sanjuan | Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis[END_REF][START_REF] Florey | Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes[END_REF], we further sought to assess the recruitment of LC3 and LAMP-1 to macroendocytic vacuoles after AC engulfment by HPFs. Western blotting analyses revealed that HPFs co-cultured with LAC contained higher levels of the lipidated LC3 form (LC3-II) compared to control cells suggesting that LAC uptake led to LC3-II conversion in HPFs (Figure 4A). Confocal microscopy analyses of LC3 immunostaining after cell fixation showed that LC3 could be recruited to AC-containing vacuole (Figure 4B). Thus, LC3-II conversion after AC uptake might be at least partly associated to the direct LC3 lipidation onto macroendocytic vacuole. Taken together, these results indicate that AC uptake by HPFs can initiate LC3-associated phagocytosis as previously described in macrophages [START_REF] Martinez | Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells[END_REF][START_REF] Sanjuan | Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis[END_REF][START_REF] Florey | Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes[END_REF]. Furthermore, a positive staining of some macroendocytic vacuoles with Lysosomal-associated membrane protein 1 (LAMP-1), a classical marker of lysosomal compartments [START_REF] Alvarez-Dominguez | Internalized Listeria monocytogenes modulates intracellular trafficking and delays maturation of the phagosome[END_REF][START_REF] Tjelle | Phagosome dynamics and function[END_REF], suggest a fusion of AC-containing vacuoles with lysosomes (Figure 4B). Further work is necessary to establish fully the significance of these observations.

Materials and Methods:

Cell culture HeLa (HPV18 DNA-positive) and Ca Ski (HPV16 DNA-positive) cervical cancer cell lines were obtained from American Type Culture Collection (Rockville, MD, USA) and used to generate early (EAC) or late (LAC) apoptotic cells. They were maintained as previously described [START_REF] Hirchaud | Isoliquiritigenin induces caspase-dependent apoptosis via downregulation of HPV16 E6 expression in cervical cancer Ca Ski cells[END_REF].

Human primary fibroblasts (HPFs) were isolated from surgical residues which are not subject to validation from an ethics committee and patient's consent in accordance with the law L.1245-2 of the "Code de la santé publique" applied in France. However, the laboratory that provided HPFs has documents stating the patient's non-opposition to the use of surgical residues for medical research in accordance with the law L.1211-2.

HPFs were grown at 37°C (5% CO2) in complete DMEM L-glutamine (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS; Lonza) and 10 × 10 4 U/L penicillin/streptomycin (Lonza).

Human monocytes were isolated and purified from peripheral blood mononuclear cells (PBMC) of buffy coat from healthy volunteers (Etablissement Français du Sang) using Ficoll-Hypaque (GE Healthcare, Orsay, France). After isolation from PBMC using a CD14+ selection kit (Miltenyi Biotech, Paris, France), monocytes were differentiated into human monocyte-derived macrophages (Mφ) by culturing cells at 1 × 10 6 cells/mL in a 24-well plate for 7 days in RPMI (Gibco, Life Technologies) supplemented with 10% FBS (Gibco) and 50 ng/mL macrophage colony-stimulating factor (M-CSF; Miltenyi Biotech). Non-adherent cells were removed by washing prior experiments.

Cells were routinely checked for mycoplasm contamination.

Generation of apoptotic cells

Twenty-four hours prior to apoptosis induction, cancer cells were seeded at 5 × 10 Endocytosis assay. HeLa and Ca Ski cells were fluorescently labeled with PKH67 (green) prior to apoptosis induction according to the manufacturer's instructions (PKH67-Fluorescent Cell Linker Kits for General Cell Membrane Labeling; Sigma-Aldrich). In brief, 10 7 HeLa or Ca Ski cells/mL were washed in serumfree culture medium and resuspended in 2 mL PKH67-containing Diluent C (2 × 10 -6 M) for 4 min at RT. Nonlabeled HPFs and Mφ (10 4 and 10 5 cells/cm 2 respectively) were seeded on coverslips of 1.9 cm 2 in a 24-well plate. Then PKH67-labeled apoptotic cells were co-cultured with HPFs or Mφ at a 10:1 ratio for different time periods at 37°C in 500 µL DMEM according to our previous data [START_REF] Gaiffe | Apoptotic HPV positive cancer cells exhibit transforming properties[END_REF]. Coverslips were washed twice with PBS to remove the non-ingested apoptotic cells. HPFs or Mφ were fixed with 3.7% (w/v) PFA. F-actin was stained with phalloidin-tetramethylrhodamine-isothiocyanate (TRITC) (Sigma-Aldrich) (2.5 × 10 -2 g/L) for 30 min at 4°C and DNA was stained with 300 nM DAPI for 5 min at RT. Cells were finally examined and images captured as described above. For each condition, more than 200 HPFs or Mφ were randomly observed and scored by two independent blinded observers.

Uptake of apoptotic cells by HPFs was also investigated by position-referenced microscopy to track the fate of HPFs exposed to apoptotic cells, as we previously described in detail [START_REF] Galeano | Position-referenced microscopy for live cell culture monitoring[END_REF]. Briefly, (

Western blotting

Western blotting was performed as described previously [START_REF] Gaiffe | Apoptotic HPV positive cancer cells exhibit transforming properties[END_REF]. Cells were collected in RIPA lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% Na deoxycholate, 1 mM EDTA) containing anti-proteases (30 μg/mL) (Roche) and proteins were extracted from whole cells. Protein concentrations were quantified using the Biorad Protein Assay® (BioRad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions. Thirty μg of proteins were boiled in Laemmli's buffer (BioRad Laboratories) supplemented with 5% β-mercaptoethanol and run on 15% SDS-polyacrylamide gel. Proteins were then electrotransferred onto Hybond™-P PVDF membranes (Amersham Pharmacia Biotech Europe GmbH, Saclay, France), probed overnight with rabbit polyclonal anti-LC3A/B (#4108) and mouse monoclonal anti-β-actin (clone AC15, Sigma-Aldrich) diluted at 1:1000 or 1:10,000 respectively according to the manufacturer's instructions. After washing, immune complexes were revealed with horseradish peroxidase-(HRP) conjugatedgoat-anti-rabbit Ig and goat-anti-mouse Ig antibodies (BD Biosciences) for 1 h diluted at 1:8000 or 1:20,000 respectively. The reaction was visualized using ECL reagents (Pierce ECL2 Western Blotting Substrate, ThermoScientific) according to the manufacturer's instructions and images were captured with a charge-coupled device (CCD) camera of the imaging system Chemidoc XRS+ (Bio-Rad Laboratories). Band density was quantified by Image Lab software (Bio-Rad Laboratories) and normalized to the loading control (β-actin). The resulting values from three different experiments were expressed as LC3II/LC3I ratio respect to the control value and reported under the western blotting as mean values ± standard deviation (SD).

Statistical analysis

Data are expressed as means ± SD of at least nine measurements (corresponding to experiments performed independently at least three times in triplicate). P values were analyzed using the Wilcoxon test.

Statistical analysis was done using GraphPad Prism (GraphPad Software, San Diego, CA). Significant differences between the groups are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001.

Cell viability assay (supplementary method)

HPFs were seeded in 96-well plates (3000 cells/well) with 100 µL of complete medium/well 24 h prior to EDTA or chemical inhibitors of endocytosis treatment and incubated for 6 h at 37°C. Then 10 μL (5 mg/mL) of MTT (Cell Proliferation Kit I, Roche Diagnostics, Meylan, France) solution was added to each well. After 4 h, 100 µL of solubilization solution (10% SDS in 0.01 M HCl) were added to each well, and cells were incubated overnight. Absorbance at 570 nm wavelength was measured with a 665 nm reference wavelength using the scanning multiwell spectrofluorimeter EnVision 2012 Multilabel Reader (PerkinElmer, Courtaboeuf, France).

Discussion:

In vivo, the rapid and efficient clearance of dying cells is fundamental for tissue development and homeostasis as well as protection against chronic inflammation and autoimmunity. As typical professional phagocytes, macrophages play a central role in the efferocytosis process. Over the last few decades, it has become clear that epithelial cells, endothelial cells, smooth muscle cells, and fibroblasts, namely nonprofessional or amateur phagocytes may contribute to dying cell clearance [START_REF] Flannagan | The cell biology of phagocytosis[END_REF][START_REF] Underhill | Information processing during phagocytosis[END_REF][START_REF] Dini | Phagocytosis of apoptotic bodies by liver endothelial cells[END_REF][START_REF] Hall | Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin[END_REF][START_REF] Monks | Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release[END_REF][START_REF] Patel | Recognition of apoptotic cells by epithelial cells: conserved versus tissue-specific signaling responses[END_REF][START_REF] Wagner | Phagocytosis of dying tumor cells by human peritoneal mesothelial cells[END_REF][START_REF] Viegas | Comparison of the kinetics of maturation of phagosomes containing apoptotic cells and IgG-opsonized particles[END_REF][START_REF] Parnaik | Differences between the clearance of apoptotic cells by professional and non-professional phagocytes[END_REF]. With regard to fibroblasts, they are known to be involved in a variety of physiological (e.g. deposition of extracellular matrix, regulation of epithelial differentiation) or pathological processes (e.g. regulation of inflammation, engagement in wound healing) [START_REF] Driskell | Understanding fibroblast heterogeneity in the skin[END_REF][START_REF] Kalluri | Fibroblasts in cancer[END_REF][START_REF] Parsonage | A stromal address code defined by fibroblasts[END_REF][START_REF] Tomasek | Myofibroblasts and mechano-regulation of connective tissue remodelling[END_REF][START_REF] Chiarugi | Cancer-associated fibroblasts and macrophages: Friendly conspirators for malignancy[END_REF][START_REF] Madar | Cancer associated fibroblasts'--more than meets the eye[END_REF]. They are also able to remove apoptotic corpses [START_REF] Hall | Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin[END_REF][START_REF] Gaiffe | Apoptotic HPV positive cancer cells exhibit transforming properties[END_REF][START_REF] Holmgren | Horizontal transfer of DNA by the uptake of apoptotic bodies[END_REF]. Professional phagocytes possess a battery of apoptotic-cell surface ligands and intermediate molecules that can lead to uptake of apoptotic cells (AC). Although amateur phagocytes like fibroblasts contribute to AC efferocytosis, mechanisms are not well understood.

We have recently demonstrated that horizontal DNA transfer can occur through the uptake of apoptotic HPV positive cancer cells by human primary fibroblasts (HPFs) leading to their transformation [START_REF] Gaiffe | Apoptotic HPV positive cancer cells exhibit transforming properties[END_REF]. In the current study, by using the same in vitro co-culture approach, we confirm that HPFs take up late apoptotic cancer cells and we demonstrate that internalization depends to a significant extent on BAI1. Interestingly, HPFs discriminate precisely between viable (data not shown), dying apoptotic targets (EAC) and dead cells (LAC) and mainly engulf LAC and not dying whole cells in contrast to professional phagocytes as macrophages [START_REF] Cocco | Distinct modes of macrophage recognition for apoptotic and necrotic cells are not specified exclusively by phosphatidylserine exposure[END_REF][START_REF] Xu | IL-10-producing macrophages preferentially clear early apoptotic cells[END_REF]. This is in line with observations having reported that other amateur phagocytes also preferentially engulf apoptotic bodies [START_REF] Dini | Phagocytosis of apoptotic bodies by liver endothelial cells[END_REF][START_REF] Monks | Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release[END_REF][START_REF] Patel | Recognition of apoptotic cells by epithelial cells: conserved versus tissue-specific signaling responses[END_REF][START_REF] Wagner | Phagocytosis of dying tumor cells by human peritoneal mesothelial cells[END_REF][START_REF] Parnaik | Differences between the clearance of apoptotic cells by professional and non-professional phagocytes[END_REF]. As reported earlier [START_REF] Patel | Recognition of apoptotic cells by epithelial cells: conserved versus tissue-specific signaling responses[END_REF][START_REF] Wagner | Phagocytosis of dying tumor cells by human peritoneal mesothelial cells[END_REF][START_REF] Parnaik | Differences between the clearance of apoptotic cells by professional and non-professional phagocytes[END_REF], the AC uptake by HPF phagocytes occurs more slowly and less efficiently than by professional phagocyte macrophages (Figure 2E). These data corroborate those obtained with endothelial and peritoneal mesothelial cells, as non-professional phagocytes, exhibiting a limited internalization potential compared to macrophages [START_REF] Monks | Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release[END_REF][START_REF] Patel | Recognition of apoptotic cells by epithelial cells: conserved versus tissue-specific signaling responses[END_REF][START_REF] Wagner | Phagocytosis of dying tumor cells by human peritoneal mesothelial cells[END_REF]. In addition, the relatively long-time sequential events observed using position-referenced microscopy, suggest that upon first tethering, AC uptake by HPFs requires a critical extended recognition and transduction period that triggers actin cytoskeleton remodeling for efferocytosis. Accordingly, this hypothesis has ever been suggested from other amateur phagocytes [START_REF] Monks | Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release[END_REF][START_REF] Parnaik | Differences between the clearance of apoptotic cells by professional and non-professional phagocytes[END_REF].

To our knowledge, the molecular mechanisms underlying this delay remain elusive.

Confocal microscopy analyses reveal that the internalization process involves the typical sequence of phagosome formation (cup-shaped invaginations of the plasma membrane, that subsequently close at their distal margins to form intracellular membrane-bounded organelles) largely mediated by an active actin cytoskeleton remodeling (Figure 2F) even if macropinocytosis cannot be excluded as observed by specific restructuring of the actin cytoskeleton such as membrane ruffles (Figure 2Fc) [START_REF] Swanson | Shaping cups into phagosomes and macropinosomes[END_REF][START_REF] Krysko | Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells[END_REF]. Indeed the partial inhibition of AC uptake by dimethylamiloride (Figure 3A) that affects neither phagocytosis nor clathrin or raft-dependent endocytosis suggests that macropinosomes might proceed to AC clearance which agrees with several authors [START_REF] Hoffmann | Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells[END_REF][START_REF] Xu | IL-10-producing macrophages preferentially clear early apoptotic cells[END_REF][START_REF] Krysko | Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells[END_REF][START_REF] Ogden | C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells[END_REF]. In addition, the data obtained with cytochalasin D confirm the involvement of actin driven-phagocytosis and likely -macropinocytosis in LAC internalization (Figure 3A). In macrophages typically driving phagocytosis of apoptotic bodies, it has been demonstrated that macropinocytosis could participate in AC uptake in a PtdSerdependent manner (reviewed in Ref. [START_REF] Krysko | Clearance of apoptotic and necrotic cells and its immunological consequences[END_REF]). Thus, in our model, two engulfment mechanisms, phagocytosis and macropinocytosis, might interplay for the removal of apoptotic material. Participation of clathrin-mediated endocytosis might contribute to LAC uptake as documented by the results of our chlorpromazine experiments.

However, the low reduction of LAC internalization might also be explained by side effects of that chemical on other endocytic pathways. Indeed, chlorpromazine can interfere with the formation of phagosomes or macropinosomes (reviewed in Ref. [START_REF] Dutta | Search for inhibitors of endocytosis: Intended specificity and unintended consequences[END_REF]), likely because of the clathrin-actin crosstalk [START_REF] Shen | Phagocytic receptor signaling regulates clathrin and epsin-mediated cytoskeletal remodeling during apoptotic cell engulfment in C. elegans[END_REF].

The first step to remove AC requires to distinguish viable cells from dying cells. The flipping of membrane lipids that occurs during early stages of apoptosis results in their exposure on the outer leaflet of the plasma membrane where they can be recognized by phagocytes [START_REF] Fadok | Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages[END_REF]. Here, the use of annexin V, an inhibitor for PtdSer-mediated efferocytosis, demonstrates that the exposure of PtdSer on the outer leaflet of the AC-plasma membrane is a key event for AC recognition and subsequent uptake by HPFs (Figure 3B,C). Nevertheless, EAC which also expose PtdSer (Figure 1C) remain weakly engulfed (Figure 2A,B, C, D). It seems clear that PtdSer exposure is required but not sufficient for the target cells to be engulfed by macrophages [START_REF] Cocco | Distinct modes of macrophage recognition for apoptotic and necrotic cells are not specified exclusively by phosphatidylserine exposure[END_REF][START_REF] Devitt | CD14-dependent clearance of apoptotic cells by human macrophages: the role of phosphatidylserine[END_REF][START_REF] Segawa | Constitutive exposure of phosphatidylserine on viable cells[END_REF]. As suggested by Borisenko et al., levels of exposed PtdSer above the phagocyte sensitivity threshold might be necessary to allow an efficient apoptotic cell engulfment [START_REF] Borisenko | Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells--existence of a threshold[END_REF]. When the plasma membrane of AC becomes permeabilized (i.e., in late apoptotic cells), additional molecules are revealed to shape a new combination of 'eatme' and 'don't-eat-me' signals [START_REF] Poon | Molecular mechanisms of late apoptotic/necrotic cell clearance[END_REF], which might favor an optimal apoptotic cell engulfment by HPFs.

Our data also demonstrate that engulfment of apoptotic cancer cells is Ca 2+ -dependent, since this process is inhibited following the chelation of extracellular Ca 2+ by EDTA. They also indicate that engulfment is significantly inhibited at 4°C. It is noteworthy that these two conditions were reported to inhibit AC-tethering step that subsequently prevent AC uptake by macrophages [START_REF] Dransfield | Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells[END_REF]. Since integrins are known to be inactive at 4°C, we may suggest a role for these cell adhesion receptors in the tethering of apoptotic cells by our amateur phagocytes likely by the MFG-E8-TSP1-αvß3 or αvß5 through indirect interaction with PtdSer [START_REF] Hall | Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin[END_REF][START_REF] Hanayama | Identification of a factor that links apoptotic cells to phagocytes[END_REF][START_REF] Borisenko | Milk fat globule epidermal growth factor 8 (MFG-E8) binds to oxidized phosphatidylserine: implications for macrophage clearance of apoptotic cells[END_REF].

In 2007, TIM-4 and BAI1 have been identified in macrophages as direct phagocytic receptors for PtdSerexposing apoptotic corpses [START_REF] Kobayashi | TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells[END_REF][START_REF] Park | BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module[END_REF]. Interestingly, our data demonstrate here that BAI1 receptor was expressed by both HPFs and macrophages contrary to TIM-4 which expression is restricted to macrophages (Figure D).

Moreover, siRNA-mediated knockdown of BAI1 in our HPFs cause a significant reduction of BAI1 mRNA associated with a reduction of efferocytosis ability (Figure 3H), as already shown in a model of astrocytes [START_REF] Park | BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module[END_REF].

Together, the present results highlight the involvement of PtdSer recognition in AC efferocytosis and the role of BAI1 as a key PtdSer receptor in our amateur phagocytes. The actin cytoskeletal remodeling observed by confocal microscopy during LAC engulfment (Figure 4F) is likely the consequence of an activation of the ELMO/Dock180/Rac1 pathway by interaction with the intracellular domain of BAI1 [START_REF] Park | BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module[END_REF]. However, our experiments highlight that both PtdSer-masking and BAI1 siRNA lead to partial reduction of AC clearance, suggesting that recognition of apoptotic cells by HPFs may also be triggered by other molecules. One of the potentially involved molecules may be the fibroblast vitronectin receptor [START_REF] Hall | Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin[END_REF]. While vitronectin receptor is defined as an integrin receptor triggering thrombospondin-mediated clearance of AC [START_REF] Hall | Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin[END_REF][START_REF] Savill | Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis[END_REF], it has also been reported that this phagocytic receptor is attracted to PtdSer through indirect interactions via the bridge molecules MFG-E8 [START_REF] Hanayama | Identification of a factor that links apoptotic cells to phagocytes[END_REF][START_REF] Borisenko | Milk fat globule epidermal growth factor 8 (MFG-E8) binds to oxidized phosphatidylserine: implications for macrophage clearance of apoptotic cells[END_REF]. The formed protein complex has been shown to bind the ELMO/Dock180/Rac1 signaling complex. Thus BAI1 and vitronectin receptor signaling seem intimately interconnected. This apparent redundancy supports the idea of a complex recognition network that may explain why the identification of cell surface proteins involved in the removal of apoptotic cells by non-professional phagocytes has been found to be challenging.

Ingested cellular material can be degraded following maturation of macroendocytic vacuoles through fusion of early endosomes, late endosomes and lysosomes [START_REF] Zhou | Phagosome maturation during the removal of apoptotic cells: receptors lead the way[END_REF]. Here, we further evidence that maturation of AC-containing vacuole is associated with changes in the composition of their membranes as shown by the detection of LAMP-1, a specific marker of lysosomes (Figure 4B). Interestingly, LAMP-1 colocalizes with the key autophagy-related protein LC3 (Figure 4B). Florey et al. have recently uncovered a non-canonical activity of this autophagy protein that is associated with macroendocytic engulfment [START_REF] Florey | Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes[END_REF][START_REF] Florey | Autophagy proteins in macroendocytic engulfment[END_REF]. During this process, so-called LC3-associated phagocytosis (LAP), LC3 is directly lipidated onto a variety of single-membrane compartments such as AC-containing vacuoles [START_REF] Martinez | Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells[END_REF][START_REF] Florey | Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes[END_REF]. In professional phagocytes, Martinez et al. have suggested that direct LC3 recruitment onto the phagosome membrane promotes phagosome maturation and subsequent degradation of the engulfed AC [START_REF] Martinez | Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells[END_REF]. In our knowledge, it is the first time that LAP is observed in amateur phagocytes.

However its significance in the clearance of LAC and the possible altered intracellular processing, especially in the context of horizontal gene transfer, remain to be clarified. The identification of other elements of the engulfment machinery expressed by HPFs and the characterization of their function in clearance of AC are beyond the scope of our future investigations. HPFs were treated with or without increasing concentrations of cytochalasin D, DMA, chlorpromazine or nystatin at 37°C for 7 h, a time condition that mimicks 1 h of preincubation and 6 h of co-culture with apoptotic cells. Based on MTT assay, these data (means ± SD) represent the relative viability against the control (untreated cells) and show that the drug-dependent effect on late apoptotic cell uptake by HPFs is independent of cytotoxic effect.

  (and 6-)carboxyfluoresceine diacetate succinimidyl ester-(CFDA, SE; Invitrogen) labeled apoptotic cells were incubated with PKH26-labeled HPFs for 11 h with an observation every hour. Images recorded at different instants in time are superimposed in a common coordinate system with subpixel accuracy.Fluorescent staining. Fluorescence analyses were performed as described above using Cell Proliferation Dye eFluor® 450 (eBioscience, San Diego, CA, USA), phalloidin-TRITC, Rabbit monoclonal anti-BAI1 [SC-66815 _ Anti-Mouse, Rat, Human, BAI-1 (H270), Santa Cruz Biotechnology, Inc., Dallas, Texas, USA], rabbit polyclonal anti-LC3A/B (#4108, Cell Signalling Technology, Danvers, MA, USA), mouse anti-LAMP1 (CD107a, BD Pharmingen, BD Biosciences, Le Pont de Claix, France) primary antibodies, and DAPI according to the manufacturer's recommendations. For immunostaining, primary antibodies and goat-anti-rabbit-Alexa488and donkey-anti-mouse-Cy™3-(Jackson ImmunoResearch Laboratories, Inc, West Grove, PA, USA) conjugated secondary antibodies were used diluted at 1:100 and 1:250 respectively.Flow cytometryApoptosis detection. For sub-G1 analysis, cells were fixed overnight in ice-cold 70% ethanol. After washing with PBS, cells were incubated with propidium iodide (PI; BD Pharmingen) (10 μg/mL) in PBS supplemented with DNase-free RNase A (Thermo Fisher Scientific, Waltham, USA) (1 mg/mL) in darkness for 15 min at RT. The intracellular PI fluorescence intensities of 20,000 cells were measured for each sample using the FC500 flow cytometer (Beckman Coulter, Orange County, CA, USA). The cytometry analyses were performed using CXP software (Beckman Coulter). For the annexin V/PI apoptosis assay, the cells were stained with fluorescein isothiocyanate (FITC) annexin V from Apoptosis Detection Kit I (BD Pharmingen) and PI in darkness for 15 min at RT, resuspended in binding buffer and analyzed by flow cytometry according to the manufacturer's instructions.Endocytosis assay. HeLa and Ca Ski cells were fluorescently labeled with PKH67 (green) prior to apoptosis and HPFs were labeled with PKH26 (red) according to the manufacturer's instructions (PKH26-Fluorescent Cell Linker Kits for General Cell Membrane Labeling; Sigma-Aldrich). HPFs were seeded at 10 4 cells/cm 2 in a 24-well plate. Then PKH67-labeled HeLa or Ca Ski were co-cultured with 20,000 PKH26-labeled adherent HPFs at a 10:1 ratio for different time periods in the conditions described previously[START_REF] Gaiffe | Apoptotic HPV positive cancer cells exhibit transforming properties[END_REF]. HPFs incubated with apoptotic cells were harvested by trypsinization and engulfment was assayed using 2-color flow cytometry. The percentage of PKH26-positive cells stained positive for PKH67 was considered as the proportion of HPFs having ingested apoptotic cells. Appropriate controls were performed to set the cytometer for each color. When indicated, culture or co-culture conditions were modified prior to endocytosis assays. A PtdSer masking assay was performed to assess the contribution of the PtdSer exposed by LAC in their uptake by HPFs.Briefly, late apoptotic cells (LAC) were pre-incubated in binding buffer with 40 or 160 µg/mL unlabeled purified recombinant annexin V (BD Pharmingen) at RT for 30 min, before being used in flow cytometry endocytosis assays. To quantify the saturation rate of annexin V specific binding sites, a fraction of preincubated cells were incubated with phycoerythrin (PE) annexin V (BD Pharmingen) at RT for 30 min. Titration of blocked annexin V binding sites was performed by flow cytometry analysis. HPFs were pre-incubated with Ethylene diamine tetraacetic acid (EDTA) disodium salt (Lonza) (up to 2 mM), cytochalasin D (Sigma-Aldrich) (up to 4 µM), 5-(N,N-dimethyl)amiloride hydrochloride (DMA; Sigma-Aldrich) (up to 80 µM), chlorpromazine (Sigma-Aldrich) (up to 20 µM) and nystatin (Sigma-Aldrich) (up to 20 µM) at 37°C for 1 h before performing flow cytometry endocytosis assays at 37°C[START_REF] Ivanov | Pharmacological inhibition of endocytic pathways: is it specific enough to be useful?[END_REF]. In additional experiments, HPFs were co-incubated with apoptotic targets at 4°C. Moreover, BAI1 siRNA (On-TargetPlus SMART pool; Dharmacon, Lafayette, CO, USA) and siRNA negative control (Eurogentec, Angers, France) were introduced into HPFs by transfection (Lipofectamine® 2000; Life Technologies) 48 h before exposure to LAC. In brief, 50 µM siRNA were added to 2 × 10 4 cells incubated in Opti-MEM (Life Technologies). RNA was prepared to assess the level of BAI1 expression by Real-time RT-PCR (RT-qPCR) analysis.RNA isolation and real-time RT-PCR analysisRNA was extracted from 10 6 cells using the QIAamp® RNA Blood Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations. Contaminant genomic DNA was removed by a treatment with 0.2 U/μL of DNase I Amplification Grade (Invitrogen, Cergy Pontoise, France) before reverse transcription with 500 ng of RNA using the SuperscriptVILO cDNA Synthesis Kit (Life Technologies).Quantitation of BAI1, TIM-4, and human β-2-microglobulin (B2M) transcripts was performed with a 7500 Real Time PCR System (Applied Biosystems, Life Technologies) in the 1X TaqMan Gene Expression Master Mix (Applied Biosystems), using respectively the Hs01105174_m1 and Hs00293316_m1 Assays-onprimer: CAATCCAAATGCGGCATCT, Eurogentec) and 200 nM of probe (6-FAM-CCT-CCA-TGA-TGC-TGC-TTA-CAT-GTC-TCG-1TC-CC-BHQ-1, Eurogentec) targeting the B2M. RT-qPCR were performed in 20 µL of a reaction mixture containing 4 µL of diluted cDNA (1:5) with 45 amplification cycles (95°C for 15 sec, 60°C for 1 min). Relative quantitation of mRNA was performed according to the 2 -ΔΔCt method algorithm with B2M serving as housekeeping control and 5 serial dilutions (1:10) cDNA from Mφ as calibrator.

Figure 1 :

 1 Figure 1: Generation of early and late apoptotic cancer cells. HeLa and Ca Ski cells were exposed to 20 mJ/cm 2 dose of UV irradiation and 300 nM staurosporine for 6 or 48 h. (A) For Sub-G1 analysis, cells were fixed, washed and incubated with PI for 15 min. The intracellular PI fluorescence intensities of 20,000 cells were measured in each sample using flow cytometry. Data shown are representative of 3 independent assays in triplicate. (B) Nuclear fragmentation was observed by fluorescent microscopy after DAPI (blue) staining. All panels were photographed at X40 and scaled to the same size. Fluorescent microscopic images are representative of 3 independent assays. Bar = 30 µm. (C) Early apoptosis and late apoptosis were respectively assessed by a simple cell staining with annexin V and a double cell staining with annexin V and PI detected by flow cytometry. The ratio of EAC and LAC (%) are indicated in the given quadrant. Data shown are representative of 3 independent assays in triplicate.

Figure 2 :

 2 Figure 2: Uptake of apoptotic cells by HPFs occurs in a time-and stage-dependent manner. (A) The binding and uptake of PKH67-labeled HeLa EAC and LAC (green) by HPFs (left and middle panel respectively) or Mφ (right panel) was investigated by confocal microscopy after incubation for 6 h at 37°C. Nonlabeled HPFs or Mφ were co-cultured with PKH67-labeled apoptotic cells (AC) at a phagocyte cell/target ratio of 1:10, followed by washing to remove the non-ingested apoptotic cells. The nuclei and actin cytoskeletons of fixed HPFs or Mφ and apoptotic targets were respectively stained with DAPI (blue) and TRITC-conjugated phalloidin (red). All panels were photographed at a X40 magnification and scaled to the same size. Fluorescent microscopic images are representative of 3 independent assays. Bar = 60 µm. (B) Based on the fluorescent microscopic analyses after 2 h and 6 h of co-culture, more than 200 HPFs were scored as described in "Material and Methods" to determine the percentage of engulfed HeLa AC-(left panel) or Ca Ski AC-(right panel) phagocytosing (means ± SD). (C) For analysis by flow cytometry, PKH67-labeled HeLa EAC and LAC (green)and PKH26-labeled (red) adherent HPFs were cultured separately (upper row) or in a 10:1 ratio for 6 h at 37°C (lower row). Flow cytometry acquisition was performed as described in "Material and Methods". Co-culture assays show that HPFs became positive for PKH67. The percentage of HPFs that were phagocytosing was determined as described in "Material and Methods". Representative dot plots are shown for HPFs and HeLa AC. (D) As depicted in C, quantitative analyses (means ± SD) of apoptotic HeLa cell (left panel) or Ca Ski cell (right panel) phagocytosis by HPFs after 2 and 72 h of co-culture at a 10:1 ratio were performed from 3 experiments in triplicate. (E) Uptake kinetics of LAC by HPFs and Mφ were investigated by confocal microscopy starting from 2 h of co-culture and phagocytosis was evaluated as depicted in B. (F) The morphological analysis of the uptake of HeLa LAC by HPFs was investigated by confocal microscopy as depicted in A. All panels were photographed at a X60 magnification and scaled to the same size. Bar = 10 µm. (G) To illustrate engulfment of a HeLa LAC (arrow) by HPFs, a sequence of images were obtained by position-referenced microscopy from a co-culture between CFSE-labeled (green) HeLa LAC and PKH26-labeled (red) adherent HPFs observed at time intervals of one hour over 11 h. A time-lapse version of G is given in the Supplemental Figure 1.

Figure 3 :

 3 Figure 3: Involvement of phosphatidylserines and BAI1 receptor in the uptake of late apoptotic cells by HPFs.(A) PKH26-labeled HPFs were incubated at 4°C or preincubated or not with EDTA, cytochalasin D, DMA, chlorpromazine or nystatin at 37°C for 1 h before the co-culture with PKH67-labeled apoptotic cells for 6 h at 4°C or 37°C. Uptake was quantified by flow cytometry as depicted in Figure2C. These data (means ± SD) represent the relative uptake against the control (untreated cells). (B) PKH67-labeled apoptotic or viable cells were preincubated with or without unlabeled annexin V at RT for 30 min before incubation with PE annexin V in darkness at RT for 20 min and cytometry analysis. Data show the dose-dependent PtdSer masking of HeLa (left panel) or Ca Ski (right panel) late apoptotic cells by annexin V preincubation. The graphs are representative of 3 independent experiments in triplicate. (C) PKH67-labeled apoptotic cells were preincubated with or without unlabeled annexin V at RT for 20 min before the co-culture with PKH26-labeled HPFs. Based on cytometry analyses, the data (means ± SD) represent the relative uptake against the control (untreated cells) and show the inhibitory effect of PtdSer masking on HeLa (left panel) or Ca Ski (right panel) apoptotic cell engulfment. (D) The relative expression of BAI1 and TIM-4 PtdSer mRNA in HPFs and Mφ was assessed by RT-qPCR. (E) For analysis of the subcellular localization of BAI1 by confocal microscopy, HPFs were cultured separately (upper row). Mφ that demonstrate strong membranous staining for BAI1 were used as positive control cells (lower row). Indirect immunofluorescence studies using anti-BAI1 antibody (H270; Alexa Fluor 488 signal, green), TRITC-conjugated phalloidin staining of F-actin (red) and DAPI staining (blue) were performed. All panels were photographed at a X40 magnification and scaled to the same size. Bar = 50 µm. (F) HPFs were cultured with PKH67-labeled HeLa LAC (green) in a 1:10 ratio for 6 h at 37°C. Indirect immunofluorescence studies using anti-BAI1 antibody (H270; Cy3 signal, red) and DAPI staining (blue) were performed. All panels were photographed at a X60 magnification and scaled to the same size. Bar = 30 µm. (G) (H) PKH26-labeled HPFs were transfected with BAI1 siRNA or control siRNA. Two days after siRNA transfection, (G) relative RNA quantification of the relative BAI1 contents in siRNA-treated cells were performed, and PKH26-labeled HPFs were co-cultured with late apoptotic HeLa or Ca Ski cells. (H) Based on cytometry analyses, the data (means ± SD) represent the relative uptake of siRNA BAI1 and siRNA control transfected HPFs against the control (untreated HPFs) and show that HPFs transfected with BAI1 siRNA demonstrated a lower ability to internalize PKH67-labelled LAC compared to the control.

Figure 4 :

 4 Figure 4: The uptake of late apoptotic cells by HPFs induces LC3 lipidation onto the phagocytic vacuoles. (A) Immunoblot of LC3 and beta actin was performed to evaluate LC3 lipidation in HPFs after 24 h co-culture at a ratio of 10:1 at 37°C with LAC HeLa and Ca Ski. HPFs were used as controls. The values of the densitometric analysis were normalized and expressed as fold increase. (B) The colocalisation of LC3 (Alexa Fluor 488 signal, green) and LAMP1 (Cy3 signal, red) to the membrane of Cell Proliferation Dye eFluor® 450-labeled (violet) HeLa LAC -containing vacuole in HPFs after 24 h of co-culture at a ratio of 10:1 at 37°C was analyzed by confocal microscopy. HPF images were captured as controls. All panels were photographed at a X40 magnification and scaled to the same size. Fluorescent microscopic images are representative of 3 independent assays. Bar = 20 µm. (C) The graphs display the colocalisation of the fluorescence signals, given in arbitrary fluorescence units (AFU), for the HeLa LAC (violet AFU) and the phagosome membrane proteins (LC3, green AFU; LAMP1, red AFU). The white bars in the pictures show the localization of the cross-section corresponding to the fluorescence diagram within the HPFs. Bar = 20 µm.

Figure S1 :

 S1 Figure S1: HPF delays ingesting late apoptotic cell for hours after recognizing it. The video combines a sequence of images -obtained by confocal fluorescence microscopy and phase contrast imaging -performed from a co-culture between CFSE-labeled (green) HeLa LAC and PKH26-labeled (red) adherent HPFs observed at time intervals of one hour over 11 h.

Figure S2 :

 S2 Figure S2: The drug-dependent effect on late apoptotic cell uptake by HPFs is independent of cytotoxic effect.

  Sigma-Aldrich) for 10 min at room temperature (RT). DNA from apoptotic cells was stained with 300 nM 4´,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Invitrogen, Life Technologies, St. Aubin, France) for 5 min at RT. After an ultimate washing with PBS, glass coverslips of 1.9 cm 2 (Dutscher, Brumath, France) were

	mounted on slides in Vectashield Mounting Media for Fluorescence (VectorLabs, Burlingame, California,
	USA). Cells were finally examined by fluorescent confocal microscopy using the Olympus FluoView 1000
	(Olympus, Tokyo, Japan) with appropriate filter settings and 40X/1.0 or 100X/1.0 numeric aperture oil
	objectives. Images were captured and data were generated using the Olympus FluoView10-ASW (Version 4.2)
	software.

4

cells/cm 2 and apoptosis was induced with UV-B irradiation (UV cross-linker, Ultra-Violet Products Ltd., Cambridge, UK) (20 mJ/cm²) followed by 300 nM staurosporine (STS; Sigma-Aldrich, St. Quentin Fallavier, France) treatment for the indicated time periods. Cells (viable and apoptotic) were then washed twice thoroughly with Phosphate Buffered Saline (PBS; Lonza) before apoptosis assays and endocytosis assays described thereafter.

Confocal microscopy

Apoptosis detection. For imaging analysis, apoptotic cells were dropped on polysine® microscope slides (ThermoScientific, Illkirch, France), washed with PBS and fixed with 3.7% (w/v) paraformaldehyde (PFA;

Acknowledgments:

We thank Gwenael Rolin, Marion Tissot, Mélanie Couturier for kindly providing human primary fibroblasts and human monocytes-derived macrophages. Authors are grateful to Virginie Mougey (Plateau technique Imagerie Cellulaire et Tissulaire, FED4234, Besançon), Francis Bonnefoy, Julie Frejaville, Sylvie Fauconnet, Aurélie Baguet, Oliver Florey (Babraham Institut, Cambridge) for their technical help and advice. We thank Fiona Ecarnot for her editorial assistance.