Elma Lima Leite

Arthur Gautron

Martine Deplanche

Aurelie Nicolas

Jordane Ossemond

Minh Thu Nguyen

Fillipe L R Do Carmo

David Gilot

Vasco Azevedo

Friedrich Goetz

Yves Le Loir

Michael Otto

Dr Nadia Berkova
email: nadejda.berkova@inra.fr

Involvement of caspase-1 in inflammasomes activation and bacterial clearance in S. aureus-infected osteoblasts-like MG-63 cells

Keywords: Staphylococcus aureus, inflammasomes, caspase-1, CRISPR-Cas9 gene editing, bacterial clearance

Staphylococcus aureus, a versatile Gram-positive bacterium, is the main cause of bone and joint infections (BJI), which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1β and IL-18. The role of inflammasomes and caspase-1 in the secretion of mature IL-1β and in the defence of S. aureus-infected osteoblasts has not yet been fully investigated. We show here that S. aureus-infected osteoblast-like MG-63 but not caspase-1 knock-out CASP1 -/-MG-63 cells, which were generated using CRISPR-Cas9 technology, activate the inflammasome as monitored by the release of mature IL-1β. The effect was strain-dependent. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes-related IL-1β production. Furthermore, we found that the lack of caspase-1 in CASP1 -/-MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1 -/-MG-63 compared to wild-type cells.

Our results demonstrate that osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase-1 in bacterial clearance.

Introduction

Staphylococcus aureus is a highly adaptive and versatile Gram-positive bacterium that has major importance for human and animal health [START_REF] Liu | Molecular pathogenesis of Staphylococcus aureus infection[END_REF]. S. aureus causes diseases ranging from minor skin infections to life-threatening infections such as bacteremia, pneumonia, meningitis, endocarditis and sepsis [START_REF] Tong | Staphylococcus aureus Infections: epidemiology, pathophysiology, clinical manifestations, and management[END_REF]. S. aureus is also the main cause of bone and joint infections (BJI), particularly in the presence of orthopedic devices [START_REF] Kahl | Clinical Significance and Pathogenesis of Staphylococcal Small Colony Variants in Persistent Infections[END_REF]. Despite adequate antimicrobial therapy BJI are linked to a high percentage of relapse, often leading to the development of a chronic disease demanding particularly challenging treatments [START_REF] Del Pozo | Infection associated with prosthetic Joints[END_REF]. While research into treatment options has been focused mostly on antibacterial strategies, it is widely believed that treatement of recurrent S. aureus infections would also benefit considerably from the reinforcement of host defenses [START_REF] Valour | Antimicrobial Activity against Intraosteoblastic Staphylococcus aureus[END_REF], and a deeper understanding of the immune response is crucial for the development of new anti-infective strategies.

The innate immune response plays a crucial role in the defense against pathogens and is initiated through pattern recognition receptors (PRRs), which recognize microbial pathogen-associated molecular patterns (PAMPs) and endogenous dangerassociated molecular patterns (DAMPs) [START_REF] Rivera | Innate cell communication kick-starts pathogen-specific immunity[END_REF]. This leads to the activation of host defense pathways that results in the clearance of infections. The innate immune response against microbes involves a major inflammatory pathway known as the activation of inflammasomes, multi-protein signaling complexes that assemble after recognition of danger signals and/or pathogens [START_REF] Schroder | The inflammasomes[END_REF]. Inflammasomes consist of a family of cytosolic receptors called NLRs (nucleotide binding domain and leucine rich repeats containing receptors), PYHIN protein family and enzymatic components, most notably caspase-1 (canonical inflammasomes) and less frequently caspase-11 in mice [START_REF] Broz | Inflammasomes: mechanism of assembly, regulation and signalling[END_REF] or caspase-4 and -5 in human (non-canonical inflammasomes) [START_REF] Choi | SERPINB1-mediated checkpoint of inflammatory caspase activation[END_REF].

Caspase-1 is synthesized in cells as an inactive precursor of 45 kDa that is matured into two subunits of 20 and 10 kDa after inflammasome activation [START_REF] Lamkanfi | Mechanisms and functions of inflammasomes[END_REF]. Most inflammasomes also use an adaptor molecule known as ASC (apoptosisassociated speck-like protein) [START_REF] Mariathasan | Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf[END_REF]. After stimulation with pathogens or DAMPS, inflammasome assembly leads to the autocatalytic cleavage of caspase-1, processing of pro-IL-1β and pro-IL-18 and secretion of mature IL-1β and IL-18 [START_REF] Strowig | Inflammasomes in health and disease[END_REF]. Furthermore, inflammasome activation may trigger pyroptosis, an inflammatory form of cell death. The role of inflammasomes in the innate immune response against various microbes has been intensively explored [START_REF] Vladimer | Inflammasomes and host defenses against bacterial infections[END_REF][START_REF] Higa | Lose the battle to win the war: bacterial strategies for evading host inflammasome activation[END_REF]. Special attention has been paid to the investigation of the role of inflammasomes during infection with intracellular pathogens, such as Salmonella typhimurium, Legionella pneumophila, Listeria monocytogenes [START_REF] Amer | Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf[END_REF][START_REF] Zaki | Salmonella exploits NLRP12-dependent innate immune signaling to suppress host defenses during infection[END_REF][START_REF] Maltez | Inflammasomes coordinate pyroptosis and natural killer cell cytotoxicity to clear infection by a ubiquitous environmental bacterium[END_REF][START_REF] Morales | A type I IFN-dependent DNA damage response regulates the genetic program and inflammasome activation in macrophages[END_REF]. Many studies also investigated the role of inflammasomes in various types of cells during infection with the facultative intracellular pathogen S. aureus: in phagocytes and epithelial cells [START_REF] Ma | LncRNA XIST mediates bovine mammary epithelial cell inflammatory response via NF-κB/NLRP3 inflammasome pathway[END_REF][START_REF] Kremserova | Frontline Science: Staphylococcus aureus promotes receptor-interacting protein kinase 3-and protease-dependent production of IL-1β in human neutrophils[END_REF], in human and rat conjunctival goblet cells [START_REF] Mcgilligan | Staphylococcus aureus activates the NLRP3 inflammasome in human and rat conjunctival goblet cells[END_REF], in human keratinocytes [START_REF] Simanski | The Inflammasome and the Epidermal Growth Factor Receptor (EGFR) Are Involved in the Staphylococcus aureus-Mediated Induction of IL-1alpha and IL-1beta in Human Keratinocytes[END_REF].

Additionally, it was reported that the expression of inflammasomes-associated proteins such as the sensor molecule NLRP3 and caspase-1 in infectious bone fragments from patients with osteomyelitis was significantly higher than in uninfected bone. Similarly, in S. aureus-induced murine osteomyelitis model, higher expression of these proteins was reported [START_REF] Zhu | Inhibition of pyroptosis attenuates Staphylococcus aureus -induced bone injury in traumatic osteomyelitis[END_REF]. However, the role of inflammasomes, S. aureus phenol soluble modulins (PSMs) and caspase-1 in the secretion of mature IL-1β as well as in the defence of S. aureus-infected osteoblasts has not yet been fully investigated. In this study we used human MG-63 osteoblasts-like cells to model the infection of osteoblasts in the context of S. aureus-associated bone infections [START_REF] Wright | Regulation of chemokine gene expression and secretion in Staphylococcus aureus-infected osteoblasts[END_REF][START_REF] Selan | Serratiopeptidase reduces the invasion of osteoblasts by Staphylococcus aureus[END_REF]. We opted for the establishement of CASP1 -/- MG-63 cell line using CRISPR-Cas9 system (see Experimental procedures, Selection of the cell model).

We discovered that in response to S. aureus infection, wild-type (WT) osteoblast-like MG-63 cells activate inflammasomes in contrast to CASP1 -/-MG-63 cells.

Inflammasome activation was monitored by caspase-1 activation and the release of mature soluble IL-1β. Mutant S. aureus strains were employed, demonstrating a key contribution of PSMs in triggering the release of soluble IL-1β, which is strongly implicated in the pathology of BJI [START_REF] Lee | IL-1 plays an important role in the bone metabolism under physiological conditions[END_REF]. In addition to the key role of caspase-1 in the release of pro-inflammatory cytokines in infected osteoblasts-like cells, we demonstrated that caspase-1 is implicated in cell defense mechanism such as a restriction of the intracellular replication of S. aureus that is critical in the development of the infection.

Results:

Caspase-1 activation and IL-1β release triggered by inflammasome activators in human osteoblasts-like MG-63 cells and THP-1 cells

In order to monitor inflammasome activation in the human osteoblast-like MG-63 cells the detection of active caspase-1 by Western blot analysis was set up in samples containing cells and cell supernatants. As shown in Fig. 1A, exposing MG-63 cells to LPS+ATP, two well-known inflammasomes activators, led to the detection of a 20 kDa band corresponding to the active caspase-1 p20 fragment. The activation of caspase-1 was associated with the production of IL-1β, as the exposure of cells to LPS+ATP resulted in the production of mature IL-1β 6 h post-infection (Fig. 1B). These results suggest that MG-63 cells form functional inflammasomes and that their activation can be estimated by IL-1β release.

To compare the ability of inflammasome activation by professional versus non- There are 6 isoforms of caspase-1 encoded by the CASP1 gene, located on chromosome 11 [START_REF] Feng | Caspase-1zeta, a new splice variant of the caspase-1 gene[END_REF]. Caspase-1 alpha and beta are the main transcripts in human cells. To generate CASP1 -/-MG-63 cells we used a previously validated sgRNA ATTGACTCCGTTATTCCGAA [START_REF] Schmid-Burgk | Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells[END_REF] (Fig. 2). Based on sequences analysis, the sgRNA is predicted to target four Caspase-1 isoforms including alpha and beta. Following lentiviral transduction of sgRNA and puromycine selection, individual clones, which were identified as a result of limiting dilution of puromycin-treated cells, were tested by Western blotting for the lack of the 45-kDa band corresponding to pro-caspase-1. Finally, after the cloning procedure described in Experimental procedures, three clones of CASP1 -/-MG-63 (G2, B9 and C2 clones originated from three different parental clones) were selected for further analysis.

Western blot analysis of WT MG-63 vs CASP1 -/-MG-63 G2 or CASP1 -/-MG-63 B9 or CASP1 -/-MG-63 C3 cells confirmed the lack of pro-caspase-1 in all three CASP1 -/- MG-63 clones (Fig. 2B).

S. aureus invades and persists inside human osteoblast-like MG-63 cells

We and others showed that S. aureus can be internalized and survive within professional phagocytes, such as macrophages [START_REF] Flannagan | Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination[END_REF], as well as within non phagocytic epithelial cells or osteoblasts [START_REF] Mohamed | Intracellular proliferation of S. aureus in osteoblasts and effects of rifampicin and gentamicin on S. aureus intracellular proliferation and survival[END_REF][START_REF] Deplanche | Staphylococcus aureus induces DNA damage in host cell[END_REF]. To study the capacity of intracellular S. aureus to induce inflammasomes formation in human osteoblasts-like MG-63 cells, we confirmed the presence of intracellular bacteria using transmission electron microscopy (Transmission EM) at different time points. An examination of electron micrographs of the infected MG-63 cells 45 min post infection reveals the presence of intracellular methicillin-resistant S. aureus strain MW2 (USA400), and bacteria that are in close contact with MG-63 cells surface (Fig. 3). Infected host cells treated with lysostaphin and gentamicin as described in Experimental procedures were observed from 6 h post-infection up to 12 days. The presence of only intracellular S. aureus was evidenced by Transmission EM (Fig. 3), while no extracellular bacteria were detected by CFU determination when supernatants of infected MG-63 cells treated with antibiotics at different time points (from 6h to 12 days) were analyzed. Bacteria were detected in the cytoplasm of MG-63 cells 6 hours, 3 days, and 12 days post-infection. The kinetics of the infection firstly was carried out using the hypervirulent CA-MRSA strain MW2 (Baba et al., 2002).

These experiments allowed the setting up of experimental conditions that ensure the detection of intracellular S. aureus in infected cells at the end of the incubation. In a previous work [START_REF] Bouchard | Inhibition of Staphylococcus aureus invasion into bovine mammary epithelial cells by contact with live Lactobacillus casei[END_REF], we found that the adhesion and internalization capacities of the S. aureus strain that reproducibly induce mild infection were higher than those of the more virulent strain that caused severe infection. Thus, to ensure a higher number of internalized S. aureus with an identical MOI, we used the strain SA113, a naturally agr-deficient S. aureus strain, which is reportedly less virulent than the wild type [START_REF] Iordanescu | Two restriction and modification systems in Staphylococcus aureus NCTC8325[END_REF][START_REF] Periasamy | How Staphylococcus aureus biofilms develop their characteristic structure[END_REF]. To confirm the presence of intracellular SA113 inside of the host cells CFU of SA113 recovered from WT MG-63 cells were determined as described in Experimental procedures. The results were presented as Survival rate (%) (Fig. S1). We showed that MW2 and SA113 strains had comparable survival rates (Fig. S1).We observed that 5 days postinfection the number of internalized bacterial cells was drastically decreased. A very low CFU number was found at the end of experiment.

Inflammasome is involved in the caspase-1 dependent release of IL-1β by S. aureus infected MG-63 cells

Activation of the NLRP3 inflammasome involves the oligomerization of the adaptor protein ASC (apoptosis-associated speck-like protein containing A CARD) into specklike aggregates [START_REF] Masumoto | Pyrin N-terminal homology domain-and caspase recruitment domain-dependent oligomerization of ASC[END_REF]. This structure recruits and activates procaspase-1, which promotes the maturation of IL-1β and IL-18.

To ensure that the depletion of Caspase-1 in CASP1 -/-MG-63 cells does not impair upstream events of inflammasome formation, the level of NLRP3 expression and the formation of ASC specks was analyzed in WT MG-63 and CASP1 -/-MG-63 clones.

NLRP3 mRNA expression was estimated by qPCR. As shown in Fig. 4A the exposure of MG-63 cells or CASP1 -/-MG-63 G2 clone to inflammasome inducers ATP+nigericin resulted in the increase of NLRP3 expression. NLRP3 protein expression was estimated by FACS using Alexa Fluor® 488-conjugated anti-human NLRP3 antibody and corresponding isotype control antibody. As shown in Fig. 4A, equal NLRP3 mRNA expression levels were observed in WT and CASP1 -/-MG-63 G2 cells. The exposure of both types of cells to S. aureus SA113, an agr-deficient strain widely used in staphylococcal research [START_REF] Herbert | Repair of global regulators in Staphylococcus aureus 8325 and comparative analysis with other clinical isolates[END_REF], resulted in similarly increased levels of fluorescence. Mean fluorescence intensity (MFI) increased from 2700 to 3800 in WT MG-63 cells after S. aureus infection, similarly to CASP1 -/-MG-63 G2 cells, which showed an MFI increase from 2800 to 3900 (Fig. 4B). Similar results were obtained with CASP1 -/-MG-63 B9 and CASP1 -/-MG-63 C3 clones (Fig. S2).

Taking into account that both types of cells contain NLRP3 (Fig. 4A), while procaspase-1 is present only in WT MG-63 cells (Fig. 2B), we next determined whether an exposure of both cell lines to S. aureus SA113 led to the formation of ASC specks.

As shown in Fig. 5A, 6 h post-infection WT MG-63 cells form ASC speck (green arrows). An exposure of CASP1 -/-MG-63 G2 clone to S. aureus induced an even higher number of ASC speck-forming cells (green arrows). Quantification of ASC speck forming cells by immunofluorescence microscopy provide an opportunity to analyse the inflammasome activation pathway even in the absence of Caspase-1.

Consequently, we analyzed immunofluorescence staining for the adaptor ASC in WT and CASP1 -/-MG-63 cells. As shown in Fig. 5A, S. aureus infection triggered assembly of the ASC specks in the cytosols of WT as well as CASP1 -/-MG-63 G2 clone (green). The number of ASC speck-forming cells in CASP1 -/-MG-63 G2 clone was twice as high as that in WT MG-63 cells (Fig. 5B). Similar results were obtained with CASP1 -/-MG-63 B9 and CASP1 -/-MG-63 C3 clones (Fig. S3, Fig. S4A).

To understand if the observed difference in the number of ASC-forming cells depends on the amount of intracellular bacteria, CFU of S. aureus recovered from WT MG-63 cells and CASP1 -/-MG-63 clones were determined as described in Experimental procedures. A 50:1 MOI was used in this experiment. As shown in Fig. 5C,6 It was previously reported that transcripts for IL-1β and IL-18 were observed after an exposure of mice osteoblasts to S. aureus. However, secreted IL-1β and IL-18 were not detected despite the presence of active caspase-1 [START_REF] Marriott | Bacterial infection of osteoblasts induces interleukin-1beta and interleukin-18 transcription but not protein synthesis[END_REF]. To understand whether S. aureus activates inflammasomes in human osteoblast-like MG-63 cells, we measured IL-1β production in WT and CASP1 -/-MG-63 cells.

The assessment of IL-1β in WT and CASP1 -/-MG-63 cells exposed to inflammasomes activators LPS+ATP (Fig. 4C) as well as to S. aureus SA113 (Fig. 4D) revealed the lack of IL-1β production in CASP1 -/-MG-63 G2 in contrast to WT MG-63 cells.

To clarify the possible role of human osteoblasts in the production of IL-1β, we analyzed the kinetics (from 2 to 11 days) of IL-1 β production by WT and CASP1 -/-MG-63 G2 cells exposed to S. aureus SA113. As demonstrated in Fig. 6A, the level of IL-1β increased from day 2 to 11. In contrast, IL-1β was not detected in supernatants from infected CASP1 -/-MG-63 G2 cells during the tested period. Similar results were obtained with CASP1 -/-MG-63 B9 and CASP1 -/-MG-63 C3 cells (Fig. S5).

To verify whether IL-1β release by infected osteoblasts-like cells was strain-dependent, we analyzed the kinetics of IL-1β release by MG-63 cells exposed to different S. aureus strains: LAC (USA300), MW2 (USA 400) and SA113. As shown in Fig. 6B, all strains induced IL-1 β release; however, MW2 and LAC induced significantly higher levels of IL-1β than SA113. IL-1β was not detected in the supernatants of MG-63 cells exposed to killed bacteria of any of the three strains, suggesting that factors associated with viable bacteria are involved in inflammasome activation.

S. aureus PSM toxins are involved in stimulation of IL-1β release by infected MG-

cells

The quorum-sensing system in S. aureus known as the accessory gene regulator (Agr) regulates the expression of many virulence factors. Agr regulates the expression of most S. aureus toxin genes. Of note, the expression of PSMs encoding genes (PSMα 1 to 4, PSMβ 1 and 2, and δ-toxin sometimes called PSMƔ) is tightly controlled by the Agr system [START_REF] Queck | RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus[END_REF][START_REF] Otto | Basis of virulence in community-associated methicillin-resistant Staphylococcus aureus[END_REF]. It has previously been shown that PSMs stimulate the production of inflammatory cytokines by infected keratinocytes [START_REF] Syed | Staphlyococcus aureus phenol-soluble modulins stimulate the release of proinflammatory cytokines from keratinocytes and are required for induction of skin inflammation[END_REF]. Furthermore, our results on the strain dependence of IL-1β stimulation in MG-63 cells suggested a possible involvement of Agr, since the cells infected with the Agr-defective SA113 strain produced low levels of IL-1β compared to the cells infected with strains MW2 and LAC (pTX∆16), which harbor a functional Agr system. Since Agr tightly controls PSM production in S. aureus, these considerations prompted us to investigate the role of PSMs in IL-1β production by infected MG-63 cells.

First, we analyzed LAC (USA300) wild-type and its isogenic mutant LAC∆psmαβhld (S. aureus strain lacking PSMα, PSMβ and δ-toxin) for their ability to stimulate the release of IL-1β. As shown in Fig. 7A, the level of IL-1β was significantly lower in the supernatants of WT MG-63 cells exposed to LAC ∆psmαβhld compared to that in the supernatant of WT MG-63 cells exposed to wild-type LAC on days 2 and 5 postinfection. To further investigate which PSMs were involved in the stimulation of IL-1β release, we used the PSM deletion strain LAC∆psmαβhld and complemented strains, expressing either the four PSMα peptides (LAC∆psmαβhld-pTX∆α1-4), the two PSMβ peptides (LAC∆psmαβhld-pTX∆β1-2), or the δ-toxin (LAC∆psmαβhld-pTX∆hld) and monitored IL-1β levels in cell supernatants exposed to those strains up to 9 days postinfection. As shown in Fig. 7B, there was a significant decrease in the IL-1β released by cells exposed to the LAC∆psmαβhld strain compared to LAC. Exposure to complemented mutants demonstrated that the release was partially restored when the strains were complemented with PSMα (LAC∆psmαβhld-pTX∆α1-4), PSMβ (LAC∆psmαβhld-pTX∆β1-2) or δ-toxin (LAC∆psmαβhld-pTX∆hld). However, the difference was statistically significant only when strains were complemented with PSMβ (LAC∆psmαβhld-pTX∆β1-2) at days 7 and 9 or, at earlier time points (days 2 and 5), with both PSMβ and PSMα (LAC∆psmαβhld-pTX∆α1-4), (Fig. 7B). For example, the cells exposed to PTXΔ16 produce 85±10 pg/mL of IL-1β 7 days postinfection. The level of IL-1β was decresed to 40±5 pg/mL produced by the cells exposed to PTXΔαβγ and was increased to 43±5 pg/mL, 70±10 pg/mL and 40±8 pg/mL produced by the cells exposed to LAC∆psmαβhld-pTX∆α1-4, LAC∆psmαβhld-pTX∆β1-2 and LAC∆psmαβhld-pTX∆hld correspondently.

To check if the difference in IL-1β production was linked to the bacterial burden, we analyzed CFU numbers of tested strains. As shown in Fig 7C, there was no difference between the CFU numbers of all S. aureus strains used in our study 2 h post-infection.

Six hours post-infection CFU of LAC∆psmαβhld-pTX∆α1-4 was significally higher than the CFU number of LAC∆psmαβhld, while there were no differences between CFU numbers of LAC∆psmαβhld-pTX∆β1-2, LAC∆psmαβhld-pTX∆hld and LAC∆psmαβhld, showing that the greater production of IL-1β induced by LAC∆psmαβhld-pTX∆β1-2 strain did not depend on bacterial burden.

S. aureus clearance by osteoblasts-like MG-63 cells depends on caspase-1

It has been reported that caspase-1 is essential for the restriction of intracellular pathogen replication in professional phagocytes [START_REF] Amer | Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf[END_REF], however the role of caspase-1 in bacterial clearance in non-professional phagocytes, which include osteoblasts-like cells, has not been investigated. Here, we found that 6 h post infection the number of CFU was significanty higher in CASP1 -/-MG-63 cells than in WT MG-63 cells (Fig. 5). We therefore analyzed whether the lack of caspase-1 in CASP1 -/-MG-63 cells is correlated with a failure to control the intracellular replication of S. aureus bacteria. The quantity of intracellular bacteria during S. aureus infection of WT and CASP1 -/-MG-63 cells was determined after gentamicin treatment of infected cells.

According to CFU determination bacterial internalization was not impaired in CASP1 - /-MG-63 cells, as we observed equivalent levels of internalized bacteria in WT MG-63 cells 2 h post-infection, with ~ 10 2 CFU /10 5 host cells. Six and 24 hours post-infection, the number of viable bacteria recovered from infected cells was significantly higher in CASP1 -/-MG-63 than in WT MG-63 (Fig. 8A). These results suggest that the lack of caspase-1 impairs the ability of osteoblasts-like cells to restrict S. aureus growth.

These findings were further confirmed using confocal microscopy. As shown in Fig. 8B, 6 h post-infection a considerably higher number of intracellular S. aureus cells (red) was observed in CASP1 -/-MG-63 cells compared to WT MG-63 cells. This difference between the two cell types is even higher 24 h post infection. Phase contrast and fluorescent image of host cells bearing S. aureus were employed to visualize the intracellular localization of bacteria. We have presented inset boxes, which indicate the regions of the merged images that are shown at a higher magnification on the right of each image (Zoom) (Fig. 8B). As seen in Fig. 8B, mCherry expressing bacteria are localized in the host cell cytoplasm in the close vicinity to DAPI-stained nuclei. Threedimension confocal microscopy was used to confirm the cytoplasmic localization of internalized S. aureus. Z-stack (Fig. 9A) was used to obtain 3D reconstituted images of S. aureus-infected host cells (Fig. 9B). Multiple images were acquired in different focal planes to create a Z-stack of images (data) through the Z axis. As seen in Fig.

Discussion

Despite antibiotic therapy and complex surgical procedures, S. aureus BJI is particularly difficult to treat. This is partly due to the tremendous capabilities of S. aureus to form biofilm, particularly in the presence of a foreign body as the formation of biofilm in the absence of material is not so frequently documented, and small-colony variants, and to invade and persist into osteoblasts, leading to chronicity and relapse of infection [START_REF] Tuchscherr | Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection[END_REF][START_REF] Vuong | Investigational drugs to treat methicillinresistant Staphylococcus aureus[END_REF][START_REF] Trouillet-Assant | Adaptive processes of Staphylococcus aureus isolates during the progression from acute to chronic bone and joint infections in patients[END_REF].

To persist within the host cells, S. aureus also developed strategies to circumvent the host cell defenses [START_REF] Alekseeva | Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency[END_REF][START_REF] Deplanche | Phenolsoluble modulin α induces G2/M phase transition delay in eukaryotic HeLa cells[END_REF][START_REF] Deplanche | Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells[END_REF][START_REF] Deplanche | Staphylococcus aureus induces DNA damage in host cell[END_REF]. Here we aimed to investigate the contributions of internalized S. aureus on alteration of host defense events such as inflammasomes activation and bacterial clearance in infected osteoblasts. The activation of inflammasomes during infection have been extensively characterized in professional phagocytes [START_REF] Miller | Inflammasomemediated production of IL-1beta is required for neutrophil recruitment against Staphylococcus aureus in vivo[END_REF][START_REF] Lamkanfi | The Inflammasomes[END_REF][START_REF] Shimada | Staphylococcus aureus evades lysozyme-based peptidoglycan digestion that links phagocytosis, inflammasome activation, and IL-1beta secretion[END_REF][START_REF] Broz | Inflammasomes: intracellular detection of extracellular bacteria[END_REF]. The activation of inflammasomes was also demonstrated in infected osteoblasts, which play a key role in BJI [START_REF] Yoshida | PKR induces the expression of NLRP3 by regulating the NF-κB pathway in Porphyromonas gingivalisinfected osteoblasts[END_REF]. Osteoblasts synthesize various bone matrix proteins that are involved in bone formation, and that regulate the maturation of osteoclasts responsible for bone resorption [START_REF] Ruscitti | The role of IL-1β in the bone loss during rheumatic diseases[END_REF]. IL-1β, a proinflammatory cytokine, plays a pivotal role in bone metabolism [START_REF] Lee | IL-1 plays an important role in the bone metabolism under physiological conditions[END_REF]. IL-1β is mainly expressed by monocytes, macrophages, and dendritic cells [START_REF] Lopez-Castejon | Understanding the mechanism of IL-1β secretion[END_REF][START_REF] Franchi | NLRC4-driven production of IL-1β discriminates between pathogenic and commensal bacteria and promotes host intestinal defense[END_REF][START_REF] Lima-Junior | Inflammasome-derived IL-1β production induces nitric oxide-mediated resistance to Leishmania[END_REF][START_REF] Chang | Dectin-2 is a primary receptor for NLRP3 inflammasome activation in dendritic cell response to Histoplasma capsulatum[END_REF], and is processed into a soluble mature form by active caspase-1, an inflammasomes compound. In the present work, we focused on the monitoring of soluble IL-1β produced by infected osteoblasts. We employed an in vitro model of intracellular bacterial infection using human osteoblasts-like MG-63 cells infected with

S. aureus.

We demonstrated that human osteoblast-like MG-63 cells release soluble IL-1β 6 h post treatment with LPS+ATP, two inflammasome activators. IL-1β release was concommittent with the appearance of an active caspase-1 20 kDa subunit, which results from an inflammasome activation in MG-63 cells. We noted that IL-1β production in MG-63 cells started later and was much lower than IL-1β production in THP-1 cells whose level of IL-1β production was similar to those previously reported [START_REF] Grahames | Pharmacological characterization of ATP-and LPS-induced IL-1beta release in human monocytes[END_REF]. Discrepancy in the levels of soluble IL-1β released by MG-63 and THP-1 is likely related to their cell types. MG-63 are indeed non-professional phagocytes whereas PMA-treated THP-1 cells differentiate into macrophages and are professional phagocytes [START_REF] Spano | PMA withdrawal in PMA-treated monocytic THP-1 cells and subsequent retinoic acid stimulation, modulate induction of apoptosis and appearance of dendritic cells[END_REF].

We observed that an exposure of MG-63 cells to S. aureus results in the progressive increase in the level of IL-1β. It started from a very low, but significant level, 6 h post-infection to 100 pg/mL several days post-infection. This suggests that osteoblasts-like cells can play a role in the host defense mechanisms. Our finding corroborate clinical observations. Recent evaluation of the link between osteomyelitis, an inflammatory process usually associated with S. aureus infection and IL1B polymorphism, indeed indicates a potential involvement of the IL1B-511T allele, suggesting that disturbance of the IL-1 system is an important factor for osteomyelitis development [START_REF] Alves De Souza | A link between osteomyelitis and IL1RN and IL1B polymorphisms-a study in patients from Northeast Brazil[END_REF]. A determination of IL-1β concentration in the synovial fluids of spontaneously osteoarthritic porcine knees, provides a range of physiologic IL-1β concentrations that can serve as a framework for the analysis of IL-1β concentration in in vitro studies [START_REF] Mcnulty | Synovial fluid concentrations and relative potency of interleukin-1 alpha and beta in cartilage and meniscus degradation[END_REF]. Median IL-1β concentration in synovial fluids was 0.109 ng/mL with mild osteoarthritis, which is similar to the IL-1β concentrations found in our in vitro conditions.

Our results contrast with previous reports describing an increase in IL-1β RNA expression but an absence of IL-1β protein synthesis in 24 h infected mice osteoblasts [START_REF] Marriott | Bacterial infection of osteoblasts induces interleukin-1beta and interleukin-18 transcription but not protein synthesis[END_REF]. This discrepancy may result from the use of human MG-63 osteoblast-like cells in our work whereas mice primary cells were employed by Marriott and collaborators. Noteworthy, IL-1β production by normal human osteoblasts-like cells was demonstrated 24 h after stimulation with LPS [START_REF] Keeting | Evidence for interleukin-1 beta production by cultured normal human osteoblast-like cells[END_REF]. The divergence between our results and the findings described by Marriott and collaborators may also result from the different S. aureus strains used in each study.

We indeed showed that induction of IL-1β release in infected cells is strongly strain-dependent, with a 10-time lower IL-1β production in cells infected with an Agr-deficient S. aureus strain.

Taking into account the pivotal role of caspase-1 in the maturation of IL-1β, we generated a CASP1 -/-MG-63 cell line using CRISPR-Cas9 gene editing for the investigation of inflammasomes involvement during S. aureus infection in a model of BJI. Despite the fact that primary cells better resemble the natural state of the organism than immortalized cells, osteoblast-like MG-63 cells were employed for CRISPR-Cas9 gene editing because primary osteoblasts can only propagate for few generations in vitro, which is not enough for the establishement of a stable CASP1 -/-cell line. MG63 cells are cancer cells and are known to differ from primary osteoblasts, especially in phenotypes that may impact inflammasome activation. Our results show events upstream the caspase-1 maturation, such as NLRP3 expression and ASC specks formation, were observed equally in both WT MG-63 and CASP1 -/-MG-63 cells. Furthermore, we showed an absence of IL-1β release following the stimulation with inflammasomes activators in CASP1 -/-MG-63 cells. We therefore consider that comparison of MG-63 cell line with CASP1 -/-MG-63 cells is relevant and the optimal model to investigate the role of inflammasomes and caspase-1 as well as pathogen clearance during infection.

The lack of IL-1β production in infected CASP1 -/-MG-63 cells demonstrated a caspase-1 dependent mechanisms of IL-1β production in human osteoblast-like cells.

Our data corroborate observations of others who demonstrated caspase-1 dependent mechanism of IL-1β processing and release by mice macrophages during infection [START_REF] Raupach | Caspase-1-mediated activation of interleukin-1beta (IL-1beta) and IL-18 contributes to innate immune defenses against >Salmonella enterica</i> serovar Typhimurium infection[END_REF]. production by infected osteoblasts appears to be particularly dependent on PSMβ among PSMs. This is noteworthy, as no specific phenotypes has been attributed to those PSMs so far. It was shown previously that PSMs induce IL-1β and IL-18 in human keratynocytes [START_REF] Syed | Staphlyococcus aureus phenol-soluble modulins stimulate the release of proinflammatory cytokines from keratinocytes and are required for induction of skin inflammation[END_REF]. In the present work, we detected S. aureus in intracellular vesicles and in the cytosol of infected cells (see Fig. 4 for MW2 and Fig. 8 for SA113). It is generally accepted that the capacity to survive inside osteoblasts is due to the release of S. aureus from the vesicles to the cytoplasm that allows S. aureus escaping the proteolytic activity of the lysosome [START_REF] Josse | Staphylococcus aureus vs. Osteoblast: Relationship and Consequences in Osteomyelitis[END_REF]. Giese et al. showed that PSMβ rather than PSMα is responsible for the efficient escape of bacteria from phagosomes [START_REF] Giese | Expression of δ-toxin by Staphylococcus aureus mediates escape from phago-endosomes of human epithelial and endothelial cells in the presence of β-toxin[END_REF]. These latter observations suggest that after S. aureus internalization PSMs promote vacuolar rupture and induce the inflammasome activation resulting in the synthesis of inflammasome-associated protein such as IL-1β in infected MG-63 cells. In addition to PSMs, the action of other factors cannot be excluded since complemented mutants did not entirely restore the level of secreted IL-1β.

The role of caspase-1 in the production of inflammatory cytokines has been extensively investigated [START_REF] Franchi | NLRC4-driven production of IL-1β discriminates between pathogenic and commensal bacteria and promotes host intestinal defense[END_REF][START_REF] Sollberger | Caspase-1: the inflammasome and beyond[END_REF], while much less attention has been paid to its impact on other immune processes. Nevertheless, many investigations point out that in addition to the processing of IL-1β and IL-18, caspase-1 regulates unconventional protein secretion [START_REF] Keller | Active caspase-1 is a regulator of unconventional protein secretion[END_REF], activates lipid metabolic pathway [START_REF] Gurcel | Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival[END_REF], restricts pathogens replication in professional phagocytes [START_REF] Master | Mycobacterium tuberculosis prevents inflammasome activation[END_REF][START_REF] Akhter | Caspase-7 activation by the Nlrc4/Ipaf inflammasome restricts Legionella pneumophila infection[END_REF] and induces pyroptosis, a proinflammatory cell death [START_REF] Fernandes-Alnemri | The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation[END_REF].

Our investigation of host defense event during S. aureus infection of non-professional phagocytes, such as the control of intracellular bacterial proliferation, reveals a drastic increase in the proliferation of internalized bacteria in osteoblastic CASP1 -/-MG-63 cells. This finding is in keeping with the previous reports demonstrating the correlation of the lack of casapase-1 activation with a failure to restrict the replication of S. aureus inside phagocytic cells [START_REF] Sokolovska | Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function[END_REF][START_REF] Cohen | S. aureus evades macrophage killing through NLRP3-dependent effects on mitochondrial trafficking[END_REF]. Indeed, it has been demonstrated that, during S. aureus infection of professional phagocytes NLRP3, inflammasomes and caspase-1 regulate phagosomes acidification that is essential for the activation of the hydrolytic enzymes required for the killing of internalized bacteria [START_REF] Ip | Phagocytosis and phagosome acidification are required for pathogen processing and MyD88dependent responses to Staphylococcus aureus[END_REF][START_REF] Sokolovska | Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function[END_REF]. Moreover, it was suggested that early activation of NLRP3 [START_REF] Ip | Phagocytosis and phagosome acidification are required for pathogen processing and MyD88dependent responses to Staphylococcus aureus[END_REF][START_REF] Sokolovska | Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function[END_REF] inflammasomes is triggered by events, such as the production of ROS, that are associated with the phagocytic process in macrophages [START_REF] Sokolovska | Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function[END_REF]. Recently, we demonstrated that S. aureus induces the production of ROS in WT MG-63 cells [START_REF] Deplanche | Staphylococcus aureus induces DNA damage in host cell[END_REF]. We hypothesize that S. aureus-induced ROS plays a similar role in inflammasome activation in non-phagocytic cells.

Collectively, our results demonstrate that human osteoblast-like MG-63 cells induce an immune response against S. aureus through inflammasomes activation and processing of IL-1β, the master cytokine of inflammation [START_REF] Dinarello | Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases[END_REF]. The outcome of the infection depends on the balance between the host immune response and the action of main S. aureus virulence factors, such as PSMs, whose production may differ among the S. aureus strains, as we have already observed in our previous work [START_REF] Deplanche | Phenolsoluble modulin α induces G2/M phase transition delay in eukaryotic HeLa cells[END_REF]. Besides our observations of inflammasomes activation, we demonstrated that CASP1 -/-MG-63 cells were unable to restrict the intracellular replication of S. aureus. It points out that the active caspase-1 prevents exacerbated intracellular replication of S. aureus in non-professional phagocytes in addition to professional phagocytes, suggesting the pivotal role of caspase-1 in S. aureus clearance independently from the type of cells. Considering this latter point, it would be interesting to estimate the possible role of different PSMs using various S. aureus wild type and PSMs mutants in the activation of Caspase-1 during long-term infection of osteoblasts. Our findings deserve further investigation to elucidate whether caspase-1 is involved in the clearance of other pathogens. Our results raise the fascinating possibility, that pathogens, which inhibit caspase-1 activation, do so not only to govern the generation of inflammatory cytokines but also to permit bacterial replication in infected cells. Current findings redefine our understanding of the role of osteoblasts in BJI, suggesting that osteoblasts, are not passive bystanders, but active players in host defenses against S. aureus infection.

Experimental procedures

Selection of the cell model.

Based on protein sequence human caspase-4 and -5 are considered as the inflammasome-associated caspases most homologous to mice caspase-11, however there is a some difference in their mechanism activation [START_REF] Viganò | Human caspase-4 and caspase-5 regulate the one-step non-canonical inflammasome activation in monocytes[END_REF].

Consequently, in order to study the inflammasomes activation during S. aureus infection in the context of BJI in human we opted for the human cells.

To silence the selected gene one can use RNA interference or the CRISPR-Cas9 gene editing approach [START_REF] Québatte | Systems-level interference strategies to decipher host factors involved in bacterial pathogen interaction: from RNAi to CRISPRi[END_REF]. Since the RNAi-mediated knockdown of expression often is not complete, the CRISPR-Cas9 editing approach was selected. Despite the fact that primary cells better reassemble the natural state of the organism than immortalized cells, osteoblast-like MG-63 cells were selected for CRISPR-Cas9 gene editing. Primary cells have a limited life span in cell culture experiments that is not sufficient for the establishment of a stable gene-deficient cell line. Moreover, freshly isolated primary cells may still have an innate mechanism to resist foreign genetic material resulting in the degradation of CRISPR components.Taking into account those points we are convinced that at present the CRISPR-Cas9 editing approach using osteoblast-like cells is the appropriate model to study the role of caspase-1 in inflammasomes activation in the context of BJI.

Maintenance of eukaryotic cells lines. The human osteoblast-like MG-63 (LGC Standards, Teddington, UK) cells and human monocyte THP1 cells (ATCC TIB-202)

were cultured in DMEM and RPMI 1640 respectively, supplemented with 10% fetal calf serum (Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C with 5% CO2 as described [START_REF] Deplanche | Staphylococcus aureus induces DNA damage in host cell[END_REF]. Trypsin/EDTA (Sigma) was used for cell subculturing to release adherent cells. In order to differentiate THP1 cells into macrophages, cells were treated with 20 nM phorbol 12-myristate 13-acetate (PMA) for 48 h [START_REF] Richter | Induction of macrophage function in human THP-1 cells Is associated with rewiring of MAPK signaling and activation of MAP3K7 (TAK1) protein kinase[END_REF].

Depletion of caspase-1 in human osteoblasts-like MG-63 using the CRISPR-Cas9

gene editing system. The caspase-1 gene editing in MG-63 cells was performed using CRISPR-Cas9 technology [START_REF] Mali | RNA-guided human genome engineering via Cas9[END_REF]. The sgRNA sequence (ATTGACTCCGTTATTCCGAA), which as previously published targets the CASP1 gene [START_REF] Schmid-Burgk | Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells[END_REF], was cloned into the lentiCRISPR v2 (Addgene #52961) [START_REF] Sanjana | Improved vectors and genome-wide libraries for CRISPR screening[END_REF]. Gene editing was achieved via lentiviral infection of MG-63 cells. Lentiviral production has been performed as recommended (http://tronolab.epfl.ch) and previously described [START_REF] Gilot | A noncoding function of TYRP1 mRNA promotes melanoma growth[END_REF] S. aureus strain description. The methicillin-resistant strains S. aureus MW2 (USA400), LAC (USA300) wild type, the isogenic mutant LAC ∆psmαβhld, which lacks the psmα and psmβ operons and in which translation of the hld gene is abolished by mutation of the start codon, LAC (pTX∆16) which carries the control plasmid, the deletion mutant LAC∆psmαβhld (pTX∆16) and the complemented strains expressing either the four PSMα peptides (LAC∆psmαβhld-pTX∆α1-4), the two PSMβ peptides (LAC∆psmαβhld-pTX∆β1-2), or the δ-toxin (LAC∆psmαβhld-pTX∆hld) were obtained from the Laboratory of Bacteriology, NIH, USA [START_REF] Wang | Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA[END_REF][START_REF] Joo | The isolation and analysis of phenol-soluble modulins of Staphylococcus epidermidis[END_REF].

The pTX∆16 plasmids were derived from plasmid pTX15 with the deletion of the xylR repressor gene. The tetracycline-resistant strains harboring plasmid pTX∆16 were grown in BHI containing 12.5 µg/mL of tetracycline. S. aureus SA113, which is an agr-deficient derivative of S. aureus strain NCTC8325 [START_REF] Iordanescu | Two restriction and modification systems in Staphylococcus aureus NCTC8325[END_REF], and SA113 (pctuf-mCherry) strain, which carries a plasmid encoding mCherry (red fluorescence) fused with the propeptide of lipase for fluorescence enhancement, were obtained from the Laboratory of Microbial Genetics, University of Tübingen [START_REF] Mauthe | WIPI-1 positive autophagosome-like vesicles entrap athogenic Staphylococcus aureus for lysosomal degradation[END_REF]. Aliquots from overnight cultures on BHI broth were diluted (1:50) in DMEM. The growth curves of mutants were similar to that of the wild type. Strains were grown at 37°C under anaerobic conditions until cultures reached an optical density of 0.6 at 600 nm, corresponding to 10 8 CFU/mL (Colony Forming Units). CFU were determined after plating a bacterial suspension on BHI agar followed by overnight incubation as described [START_REF] Alekseeva | Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency[END_REF].

Cell culture infection. 2.5x10 5 of either MG-63, CASP1 -/-MG-63, or THP1 cells were seeded in the wells of 12-well plates. Experiments were performed with PMA-treated THP1 cells. Cells then were exposed to S. aureus strains at MOI 50:1. An MOI value of 50:1 was selected in order to prevent the induction of host cell death [START_REF] Deplanche | Staphylococcus aureus induces DNA damage in host cell[END_REF]. Bacterial concentrations were estimated spectrophotometrically and were confirmed by determination of CFU. Extracellular bacteria were removed 2 h postinfection by incubating cells in cDMEM with 20 μg/mL lysostaphin and 100 μg/mL gentamicin for 2 h, which eliminates extracellular bacteria without altering intracellular bacteria [START_REF] Alekseeva | Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency[END_REF][START_REF] Deplanche | Phenolsoluble modulin α induces G2/M phase transition delay in eukaryotic HeLa cells[END_REF], followed by incubation in cDMEM containing 25 μg/mL of gentamicin. After the indicated periods adherent and floating cells were collected, centrifuged, and prepared either for Western blot analysis or for analysis by cytofluorometry.

For the determination of the number of internalized bacteria, we used the method we have previously described [START_REF] Bouchard | Inhibition of Staphylococcus aureus invasion into bovine mammary epithelial cells by contact with live Lactobacillus casei[END_REF][START_REF] Alekseeva | Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency[END_REF] Briefly, to determine the number of internalized bacteria after 2 h of infection, infected cells were lysed with 0.05% Triton X-100 in PBS, cell lysates were plated on BHI agar at different dilutions, and CFU were determined after overnight incubation as described [START_REF] Bouchard | Inhibition of Staphylococcus aureus invasion into bovine mammary epithelial cells by contact with live Lactobacillus casei[END_REF][START_REF] Alekseeva | Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency[END_REF].

To determine the intracellular survival rate of internalized S. aureus SA113 the initial internalized bacterial population was measured 2 h post-infection and was used as the starting point for intracellular survival assay and was considered as 100%. The internalized S. aureus SA113 population at the different periods was measured and expressed relative to an S. aureus SA113 intial internalization (2 h post-infection).

Gene expression analysis by qRT-PCR

Human WT MG-63 and CASP1 -/-MG-63 G2 cells were exposed to inducers of inflammasomes activation, 5 mM ATP for 30 min, following by the exposure to 5 mg/mL nigericin for 3 h. Relative NLRP3 mRNA expression was evaluated by quantitative Real-Time PCR (qRT-PCR), as described [START_REF] Deplanche | Phenolsoluble modulin α induces G2/M phase transition delay in eukaryotic HeLa cells[END_REF]. Briefly, the total RNA was isolated with a RNA II kit (Macherey Nagel) and the cDNA was synthesized using a qScript cDNA Synthesis kit (Quanta Biosiences). Reactions devoid of reverse transcriptase and reactions containing H2O instead of cDNA were used as negative controls. Primers for human NLRP3 were designed according to the sequences The relative mRNA level was calculated as × deltaCT (x = Primer efficiency).

After normalization relative quantification refers to the PCR signal of the target transcript in a treatment group divided by the values obtained from control cells arbitrarily set to 1 at the indicated period. Western blot analysis. Pro-caspase-1 and cleaved caspase-1 were detected by Western blot analysis as described [START_REF] Berkova | Aspergillus fumigatus conidia inhibit tumour necrosis factor-or staurosporine-induced apoptosis in epithelial cells[END_REF]. Briefly, 2.5x10 5 of cells were grown in 12-well plates. Pro-caspase-1 was detected in non-stimulated and stimulated cells, while active caspase-1 was detected solely in stimulated cells. Wild type (WT)

Transmission electron microscopy (Transmission EM

MG-63 or CASP1 -/-MG-63 cells were primed for 3 h with LPS (1μg/mL) (Sigma) and selected samples were stimulated with ATP (5 mM) for 15 min or after the exposure to S. aureus strains. Six hours after the beginning of the treatment cells together with cellculture supernatants were collected and diluted in RIPA lysis buffer supplemented with complete protease inhibitor cocktail, P8340 (Sigma Aldrich®). Protein samples were prepared in 5x polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer.

Proteins were separated on 12% SDS-PAGE gels and transferred onto 0.2 µM PVDF membranes. The membranes were washed three times in PBS-T (Tris-buffered saline containing 0.5% Tween 20) and blocked in 10% skim milk in PSB-T for 1 h at room temperature before being incubated with primary antibody overnight at 4°C. Caspase-1 was detected with anti-human caspase-1 antibody (1:1,000 dilution, mAb p/20, AdipoGen). Membranes were washed four times in PBS-T and incubated with HRPconjugated secondary anti-mouse IgG-HRP antibody 1:10,000 dilution, Cell Signaling Ozyme) for 1 h at room temperature, washed another four times, and incubated with Western chemiluminescent HRP substrate (ECL kit) (GE Healthcare). The image was processed using a GBOX imaging system (Syngene, Ozyme, Poitiers, France). To assess the quantity of loaded protein, membranes were re-probed with rabbit antitubuline (Cell Signaling Ozyme France) (1:1000) and bands were visualized using the ECL kit and a GBOX imaging system.

Flow cytometry analysis. For an estimation of NLRP3 protein expression in WT MG-63 vs CASP1 -/-MG-63 cells, cells were exposed to S. aureus LAC for 2 h and were fixed in 4% paraformaldehyde/PBS followed by permeabilization in 0.1% Triton/0.5% BSA/PBS. After centrifugation, cells were resuspended in 200 µl of 0.5% BSA/PBS, 5 µl of Rat Anti-Human NLRP3 Alexa Fluor® 488-conjugated Monoclonal Antibody (Catalog # IC7578G) or isotype control antibody (Catalog # IC006G) per 10 6 cells were added, and the samples were incubated for 45 min. NLRP3 expression was analyzed with an Accuri C6 flow cytometer. Data were collected from 20,000 cells, and analyzed with CFlow software (Becton Dickinson) as described [START_REF] Nguyen | Staphylococcus aureus Lpl lipoproteins delay G2/M phase transition in HeLa cells[END_REF].

Confocal microscopy. WT MG-63 or CASP1 -/-MG-63 cells were grown on cover slips and exposed to S. aureus SA113 (MOI 50:1) for 2 h. Six and 24 h post-infection, cells were fixed with 4% paraformaldehyde/PBS for 20 min, followed by permeabilisation in 0.1% triton/PBS and incubation with 20% goat serum (Sigma) as described [START_REF] Deplanche | Staphylococcus aureus induces DNA damage in host cell[END_REF]. Rabbit anti-PYCARD antibody (Coger France) diluted 1:50 in 1% BSA/PBS was applied overnight at 4°C, followed by incubation with Alexa Fluor

Statistical analysis

At least three independent assays were performed per experiment. The differences among the groups were assessed by analysis of variance (ANOVA). P-values < 0.05 were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as mean ± standard deviation (±SD).

  professional phagocytes, PMA (phorbol 12-myristate 13-acetate)-treated THP1 cells differentiated into macrophages or MG-63 osteoblast-like cells, were exposed to LPS+ATP and the level of IL-1β production was estimated at 2 h and 6 h after the beginning of the treatment. PMA-treated control THP1 cells produce ~ 7 pg/mL of IL-1β 2 h after the beginning of the treatment. Exposure of PMA-differentiated THP-1 cells to LPS+ATP resulted in a significant increase in IL-1β secretion, up to ~ 30 pg/mL, 2 h post treatment. After 6 h, PMA-treated control THP-1 cells produce ~ 10 pg/mL of IL-1β, while the level of IL-1β produced by PMA-treated THP-1 cells after an exposure to LPS+ATP was ~ 650 pg/mL. In contrast, IL-1β was not detected 2 h post-treatment while only 5±0.5 pg/mL of IL-1β was detected in MG-63 cells 6 h after the exposure to LPS+ATP. Six hours post-treatment, the level of IL-1β produced by MG-63 cells was low, but significanty above the sensitivity threshold of the detection kit, (2 pg/mL; Invitrogen ELISA kit), and dramatically contrasted with professional phagocytes PMAtreated control THP-1 cells that produce ~ 650 pg/mL of IL-1β. Establishement of CASP1 -/-MG-63 cell line using the CRISPR-Cas9 gene editing system It has been shown that stimulation is required for IL-1β transcription and protein expression by MG-63 (Koh et al., 1997), a cell line that is commonly used as a model to study BJI (Kazemzadeh-Narbat et al., 2012). To investigate the role of inflammasomes in S. aureus-infected human osteoblast-like MG-63 cells, the establishement of CASP1 -/-MG-63 cell line was carried out using the CRISPR-Cas9 gene editing system (see Experimental procedures, Selection of the cell model).

  h postinfection the number of CFU of S. aureus recovered from CASP1 -/-MG-63 G2 cells was significantly higer than those recovered from WT MG-63 cells. Similar results were obtained with CASP1 -/-MG-63 B9 and CASP1 -/-MG-63 C3 cells (Fig. S4B). Alltogether our results showing that both WT MG-63 cells and CASP1 -/-MG-63 clones, express NLRP3 and form ASC specks while only WT MG-63 cells produce IL-1β following the exposure to inflammasomes inducers or to S. aureus, approuve the monitoring of IL-1β produced by both cell lines for the analysis of inflammasomes activation.

  9A, the sections close to the bottom of the slide culture chamber exhibited the blue fluorescence of the eukaryotic cells nuclei. Ascending within this "z-stack," middle sections showed dots with intense red fluorescence, corresponding to internalized bacteria. The sections closed to the top of the slide culture chamber exhibited only blue fluorescence of DAPI-stained nuclei, and red fluorescent bacteria was not detected anymore. Compartmentalisation of S. aureus inside WT MG-63 and CASP1 -/-MG-63 cells was examined by transmission EM. Phagosomal/lysosomal membranes were visible surrounding individual bacteria or clusters of bacteria (Fig. 9, C, red arrows) together with free bacterial cells in the cytosol (Fig. 9, C, black arrows).

  The absence of IL-1β production by cells exposed to heat-killed bacteria indicates that factors associated with viable bacteria are involved in the activation of NLRP3 inflammasome. Employment of deletion and complemented PSMs mutants demonstrated that S. aureus toxins PSMs are involved in inflammasomes related IL-1β production by infected osteoblast-like cells. We would like to highlight that IL-1β

  . After antibiotic selection [puromycine (3 days, 1µg/mL)], cells were seeded in 96-well plates at 0.3 cells/well for single cell clonal expansion. Single-cell clones were tested by Western blot analysis for the presence of the 45 kDa band corresponding to pro-caspase-1. Six clones that did not present 45-kDa bands were recloned in 96-well plates (10 and 30 cells per plate). Afterwards, single-cell clones were transferred in 6-wells plate and grown to 80% of confluence. Three clones originating from three different parental clones that lacked 45-kD bands (Western Blot) were recloned in 96-well plates (10 and 30 cells per plate). All monoclones lacked 45-kD bands (Western blot). Again, three clones originated from three different parental clones were selected and grown in 25-mL flasks. CRISPR-induced genomic modifications were verified by nucleotide sequence analysis, and the lack of caspase-1 was confirmed by Western blot analysis.

  www.ncbi.nlm.nih.gov/ (NLRP3, NCBI accession number NM_001079821.2), f5'-GAAGCACCTGTTGTGCAATC-3', r5'-GCAAGATCCTGACAACATGC-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, NCBI accession # NM2046.3) f5'-CAAGGGCATCCTGGGCTAC-3', r5'-GGTGGTCCAGGGGTCTTACT-3' and PeptidylProyl Isomérase A (PPIA, NCBI accession #NM_001300981) f5'-GACCCAACACAAATGGTTCC-3', r5'-TCGAGTTGTCCACAGTCAGC-3' were used as housekeeping genes. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of an untreated control. Each 13 µl PCR mixture included 2 µl of cDNA, 200 nM of primers, 12.5 µl of IQ Sybr Green supermix (2×)(Biorad) and H2O. Amplification was carried out on a CFX96 Real Time System (Bio-Rad, Marne la Coquette, France) for 3 min at 95°C and 40 cycles of two steps consisting of 5 s at 95°C and 30 s at 60°C. The relative quantification of the mRNA levels of the target genes was determined using CFX Manager based on the deltaCT -method[START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF]. The amount of target was normalized to PPIA and GAPDH genes: deltaCT = CT (target gene) -CT (housekeeping gene) where CT represents the cycle number required to reach a defined threshold target abundance.

  488 labeled goat anti-rabbit antibody (Cell Signaling Ozyme) (1:1000) for 2 h. Cover slips were mounted with ProLong antifade containing DAPI (Vectashield, Biovalley). Confocal laser scanning microscopy (CLSM) observation of specimens was performed using a ZEISS LSM 880 inverted confocal microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with immersion objective 63× (Plan Apochromat objective, oil immersion, NA 1.4) driven by Zen software. The pinhole is set to the one Airy unit. To quantify the amount of ASC-speck forming cells the number of ASC speck out of 100 cells in the culture of WT MG-63 cells vs CASP1 -/-MG-63, G2, B9 and C2 clones was enumerated. To obtain three dimential images of host cells infected by S. aureus multiple images were acquired in different focal planes to create a Z-stack of images (data) through the Z axis. IL-1 beta quantification by ELISA. WT MG-63, CASP1 -/-MG-63 or THP1 cells were exposed either to inflammasomes inducers 1 mg/mL LPS for 30 min followed by the exposure to 5 mM ATP for 15 min (LPS+ATP) or to S. aureus strains. Undiluted cell culture supernatants were subjected to human IL-1β detection by sandwich-ELISA (Thermofischer Life Technologies, INVITROGEN 88-7261) according to the manufacturer's instructions. Briefly, wells of 96-wells plates were coated with capture anti-human IL-1β antibody and were incubated overnight at 4°C. After washing the wells were incubated in the blocking solution. Then tested samples were added to the appropriate wells. A standard curve was measured with 2-fold serial dilutions of the maximal standard concentration of 150 pg/mL. Recombinant human IL-1β was used for generating the standard curve and calibrating samples. After 2 h of incubation, biotin-conjugated anti-human IL-1β antibody was added to the wells followed by incubation with avidin-HRP for 30 min. After addition of the tetramethylbenzidine substrate solution for 15 min the reaction was stopped with stop solution and absorbance was read at 450 nm. The sensitivity of IL-1β detection is 2 pg/mL.

Acknowledgments

The authors thank Pr. Petr Broz, Department of Biochemistry, University of Lausanne, Epalinges, Switzerland for his help in design of experiments related to CRISPR editing, for invaluable suggestions in the analysis of results, for discussion and corrections of the manuscript. We thank Mary Bret for the English corrections. We thank Chantal Cauty (STLO) and Agnes Burel (MRic, Rennes University) for their precious help in performing Transmission EM. The program Germaine de Stael, PHC bi-lateral France-Switzerland collaboration (to N.B. and P.B.), French National Institute for Agricultural Research (INRA), Metaprogram GISA, LONGhealth -MPP10573 (to N.B. and M.D), the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), U.S. National Institutes of Health (NIH), project number 1 ZIA AI000904 (to M.O.), the Deutsche Forschungsgemeinschaft (DFG) SFB766 (to F.G. and M.T). E. L. L. is a recipient of a Ph.D. fellowship from Coordenação de Aperfeiçoamento de Pessoal de N´ıvel Superior (CAPES; Brazil). The ZEISS LSM880 confocal microscope was funded by the European Union (FEDER), the French Ministry of Education, Research and Innovation, the Regional Council of Brittany and INRA.

All authors reviewed the Manuscript.

Author's contribution

Conflict of Interest

The authors declare no competing financial and/or non-financial interests, all authors concur with the submission with LPS (1 μg/mL) and stimulated with ATP (5 mM) for 15 min. Six hours after the beginning of the treatment cells together with cell-culture supernatants were diluted in RIPA lysis buffer. Protein samples were prepared in 5 x SDS-PAGE sample loading buffer. Detection of 45-kDa pro-caspase-1 in MG-63 cells by Western blot analysis was performed using anti-caspase-1 antibody (AdipoGen) as described in Experimental procedures. The protein load was verified with anti-tubulin antibody. Three independent assays were performed. B, C. MG-63 or THP1 cells were incubated in 12-well plates. Afterwards, cells were primed for 2 h with LPS (1 μg/mL) and stimulated with ATP (5 mM) for 15 min. THP1 were treated with PMA before LPS+ATP treatment as described above. Two or six hours after the beginning of the treatment cell supernatants were collected, centrifuged, and the level of IL-1β was determined by a commercial sandwich-ELISA (Thermofischer Life Technologies, INVITROGEN 88-7261) as described in Experimental procedures. The sensitivity of IL-1 β detection by ELISA is 2 pg/mL. Supernatants of THP-1+LPS+ATP were diluted 1:5. The values are presented as concentrations in pg/mL. Four independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. P < 0.05 (*) and P < 0.01 (**) were considered to be significant. The values are expressed as means ± standard deviation (±SD). Protein samples were prepared in 5 x SDS-PAGE sample loading buffer. Detection of 45-kDa pro-caspase-1 in WT MG-63, CASP1 -/-MG-63 G2, CASP1 -/-MG-63 B9 and CASP1 -/-MG-63 C3 cells by Western blot analysis using anti-caspase-1 antibody (AdipoGen) was performed as described in Experimental procedures. Three independent assays were performed. A. WT MG-63 or CASP1 -/-MG-63 G2 cells were grown in 6-well plates, then were exposed to S. aureus strain SA113 at MOI 50:1 for 2 h followed by antibiotic treatment as described in Experimental procedures. After various times post-infection (6 h, 2 days, 5 days, 7, days and 11 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a sandwich-ELISA (Thermofischer Life Technologies) as described in Experimental procedures. The values are presented as concentrations in pg/mL. Three independent assays were performed. The differences among the groups were assessed by the analysis of variance (ANOVA). P-values < 0.05 ( ⃰ ) were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD).

B. WT MG-63 cells were exposed to S. aureus strains LAC (USA300, red color), MW2 (USA 400, green color), SA113 (yellow color) at MOI 50:1 or killed bacteria (violet color) for 2 h followed by antibiotic treatment as described in Experimental procedures. After various times post-infection (6 h, 2 days, 5 days, 7, days and 11 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a commercial sandwich-ELISA (Invitrogen, France) as described in Experimental procedures. The values are presented as concentrations in pg/mL. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values < 0.05 (*) were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups.

The values are expressed as means ± standard deviation (±SD). Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD). P-values < 0.05 ( ⃰ ) were considered to be significant. Three independent assays were performed.

B. MG-63 cells were exposed to USA300 LAC (pTX∆16), which carries the control plasmid, the deletion mutant LAC∆psmαβhld (pTX∆16) and the complemented strains expressing the four PSMα peptides (LAC∆psmαβhld-pTX∆α1-4), the two PSMβ peptides (LAC∆psmαβhld-pTX∆β1-2), or the δ-toxin (LAC∆psmαβhld-pTX∆hld) at MOI 50:1 for 2 h followed by antibiotic treatment as described in Experimental procedures.

After various times post-infection (2 days, 5 days, 7 days and 9 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a sandwich-ELISA (Thermofischer Life Technologies) as describe in Experimental procedures. The values are presented as concentrations in pg/mL. The differences among the groups were assessed by analysis of variance (ANOVA). Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD). Three independent assays were performed. P-values< 0.05 (*) were considered to be significant.

C. WT MG-63 or CASP1 -/-MG-63 cells were exposed to USA300 LAC (pTX∆16), which carries the control plasmid, the deletion mutant LAC∆psmαβhld (pTX∆16) and the complemented strains expressing the four PSMα peptides (LAC∆psmαβhld-pTX∆α1-4), the two PSMβ peptides (LAC∆psmαβhld-pTX∆β1-2), or the δ-toxin (LAC∆psmαβhld-pTX∆hld) at MOI 50:1 for 2 h followed by antibiotic treatment as described. Six hours post-infection cells were lysed with 0.05% Triton X-100 in PBS, cell lysates were plated on BHI agar, and CFU were determined after overnight incubation. CFU values were normalized to 10 5 host cells. Experiments were performed in triplicate. Three independent assays were performed.

Figure 8. Involvement of caspase-1 in bacterial clearance

A. WT MG-63 or CASP1 -/-MG-63 G2 cells were grown in 12-well plates overnight, then were exposed to S. aureus SA113 strain at MOI 50:1 for 2 h followed by antibiotic treatment as described. Two hours, 6 h and 24 h post-infection cells were lysed with 0.05% Triton X-100 in PBS, cell lysates were plated on BHI agar, and CFU were determined after overnight incubation. CFU values were normalized to 10 5 host cells.

Experiments were performed in triplicate. Three independent assays were performed.

The data are presented as means ± SD. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. P ˂ 0.01 (**), for the comparison of the number of internalized bacteria in CASP1 -/-MG-63 cells with those in WT MG-63 cells and P-values < 0.05 ( ⃰ ) for the comparison of the number of internalized bacteria in CASP1 -/-MG-63 cells 6 h and 24 h post-infection were considered to be significant.


B Le Daré

A Degremont

E Polard

I Morel

A Biraben

T Gicquel

Clinical and Biological Assessment of Lamotrigine and Levetiracetam Plasma Assays at the Rennes University Hospital

Keywords: antiepileptics, plasma assays, levetiracetam, lamotrigine, therapeutic management

published or not. The documents may come

Clinical and biological assessment of lamotrigine and levetiracetam plasma assays at the Rennes University Hospital

Introduction

Epilepsy is defined as a brain disorder characterized by an enduring predisposition to generate epileptic seizures and by the neurobiological, cognitive, psychological and social consequences of this condition. The definition of epilepsy thus requires the occurrence of at least one epileptic seizure [START_REF] Fisher | ILAE Official Report: A practical clinical definition of epilepsy[END_REF]. In France, the Haute Autorité de Santé (HAS) or French National Authority for Health estimates the number of people with epilepsy at 500,000 (2). The management of this pathology is mainly based on pharmacological treatment, making it possible to reduce the seizures frequency or even eliminate them.

Some antiepileptic drugs may have disadvantages in terms of their pharmacokinetic profile, including polymorphic enzymes metabolization, numerous drug interaction, active metabolites, saturable metabolism, high plasma protein binding or enzymatic inducer properties [START_REF] Patsalos | Clinical Pharmacokinetics of Levetiracetam[END_REF][START_REF] Laroche | A New Look at the Second-Generation Antiepileptic Drugs: A Decade of Experience[END_REF]. These characteristics expose patients to wide inter and intra-individual variations in plasma concentrations, thus impacting the treatment efficacy and tolerance. In order to optimize the management of patients treated with antiepileptic drugs, pharmacological follow-up may be performed under certain conditions. The indications recommended by the HAS concern: the initiation of full-dose antiepileptic treatment after a period of a few weeks; proven or suspected poor compliance; suspicion of overdose; therapeutic adjustment in the event of seizure recurrence or aggravation, drug interaction management; and specific clinical situations: pregnancy, metabolic failure, etc. (2). Despite these specific clinical situations for which an interest in the pharmacological monitoring of antiepileptics has been suggested, the data in the literature show varying results in terms of compliance with these recommendations. In a French prospective study performed in a tertiary center, 84% of 698 antiepileptic drug levels measurement had an appropriate indication according to French guidelines [START_REF] Hanin | Rates of serum level determinations of antiepileptic drugs in accord with guidelines: A clinical study at a tertiary center[END_REF]. Conversely, Affolter et al. (2009) showed in a retrospective study on 602 antiepileptic drug serum level determinations that less than half of them met the criteria for appropriateness, creating unnecessary costs [START_REF] Affolter | Appropriateness of serum level determinations of antiepileptic drugs[END_REF]. Lastly, Kozer et al. concluded in 2003 that the results of plasma antiepileptic drug assays performed in pediatric emergencies were not associated with the resulting management [START_REF] Kozer | Utility of antiepileptic drug monitoring in the pediatric emergency department[END_REF].

In view of lamotrigine and levetiracetam plasma assays increasing requests at the University Hospital of Rennes (Brittany, France), we have carried out an evaluation of these practices. The objective of this work was to evaluate the follow-up of HAS recommendations, and the impact of assay results on medical management modification.

Material and methods

This retrospective observational study was conducted on 169 patients whose samples were returned to the biological and forensic toxicology laboratory at the University Hospital of Rennes between 2015 and 2018. Two hundred and forty-two assays were analyzed: 158 of lamotrigine and 82 of levetiracetam according to the appendix.

The data were extracted from Synergy® (TD Control®) software, which provides the plasma assays results performed by tandem mass spectrometry (LC-MS/MS) at the University Hospital of Rennes.

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The variables studied concerned emergency department or hospitalization department, the molecule, the antiepileptic drug indication, the reason for prescribing the assay, the assay result, the age of the patient, and the consequences on the patient management, i.e. dosage changes, stopping or adding drugs, and the hospitalization frequency after a visit to the emergency department. These variables were collected from each patient's medical record (DxCare® Software, version 7.7.3). Considering that the time of the last intake of the drug was not indicated in these assays requests, we did not take into account the time of sampling. The consequences on medical management, analyzed by considering all the clinical situation (reason for admission in emergency department, addition or suppression of another drug, pregnancy, pathologies different from epilepsy), were compared between patients with plasma concentrations of antiepileptics in or outside therapeutic range. Based on data from the literature, the therapeutic range of 2 to 15 mg/L was selected for lamotrigine, and 6 to 40 mg/L for levetiracetam [START_REF] Schulz | Therapeutic and toxic blood concentrations of more than 800 drugs and other xenobiotics[END_REF].

Figures and statistical analyses were performed using GraphPad® software (version 5.0, La Jolla, CA, USA). The variables were described using the usual parameters, namely mean and standard deviation for quantitative variables (age and plasma concentration) and frequency for qualitative variables (molecule, indication, hospitalization frequency, and medical management changes). Some parameters were compared in percentages using a Chi² Test between the population in the therapeutic range and the population outside the therapeutic range.

Results

Characteristics of assay requests

The assay activity for levetiracetam and lamotrigine began at the Laboratory of Biological Toxicology of the University Hospital of Rennes on 11/12/15 and 29/12/15 respectively. In 2016, these two molecules represented 33 assays, compared to 136 in 2018; the activity increased 4.1-fold between these two years (Figure 1). A total of 242 assays of these two molecules were prescribed between 2015 and 2018. The mean age of the patients included in this study was 50.3 years (+/-25.4 years).

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Of all the services that used a lamotrigine and/or levetiracetam assay, the five main prescribers, who prescribed approximately 72% of the assays, were in order of frequency: emergency departments (adults, pediatrics and gynaecology), intensive care units (medical and surgical), the long-term care unit (LTCU), the neurology department, and internal medicine / infectious disease departments.

Follow-up of HAS recommendations

Of all requests for lamotrigine and levetiracetam assays between 2015 and 2018 (N=242), 207 (85.5%) were prescribed for epilepsy; only 10 (4.1%) were requested for bipolar syndromes; and 25 (10.3%) had no reasons found. Due to the lack of data on assays in patients with bipolar disorder, these were not analyzed in this study (Appendix A).

Of the 207 assays prescribed for epilepsy, 177 (85.5%) were in line with the HAS recommendations, namely: 76/177 (42.9%) as part of a therapeutic adjustment in the event of seizure recurrence or aggravation; 45/177 (25.4%) for specific clinical situations (status epilepticus, pregnancy, postpartum, or acute renal failure); 23/177 (13%) for proven or suspected poor compliance; 23/177 (13%) for suspected overdose; 8/177 (4.5%) following initiation of treatment; and 2/177 (1.1%) for drug interaction management (Figure 2).

Thirty assays (14.5%) were out of HAS recommendations, finding 10 unexplained discomfort reports, six "other" reasons (treatment follow-up after a dosage change without seizure recurrence or aggravation, and assays after stopping treatment) and 14 (46.6%) assays for routine monitoring (Figure 2). A c c e p t e d M a n u s c r i p t

Results of the assays

In the case of lamotrigine or levetiracetam treatments in epileptic patients, approximately two thirds of the assays were found in the therapeutic range (89/137 (65%) and 46/70 (65.7%) respectively). In addition, lamotrigine and levetiracetam plasma concentrations showed a higher frequency of low plasma concentrations. Taken together, these results show that the majority of assays are located in the lower therapeutic ranges (Figure 3). Depending on the reasons for prescribing antiepileptic drugs, the proportions of underdose, assays in the therapeutic range and overdose are different (Figure 4). For assays performed as part of a therapeutic adjustment in the event of seizures recurrence or aggravation, the majority of plasma concentrations were found in the therapeutic range (Figure 4A). Considering the main specific clinical situations, in cases of status epilepticus, 19/29 (65.5%) lamotrigine and levetiracetam determinations found concentrations in the therapeutic range (Figure 4B). In the assays performed during pregnancy, 8/14 (57.1%) were below the therapeutic range, of which 6/14 (42.8%) were prescribed following iterative pregnancy vomiting (Figure 4C). For assays following proven or suspected noncompliance, 14/23 (60.9%) results were under-dosed (Figure 4D). In the context of suspected overdose, the results were 1/23 (4.3%) underdose, 14/23 (60.9%) doses in the therapeutic range and 8/23 (34.8%) overdose (Figure 4E). Epilepsy treatment initiation, when following an assay, found 6/8 (75%) plasma concentrations in the therapeutic range (Figure 4F). Assays for unexplained discomfort reports found 7/8 (87.5%) plasma concentrations in the therapeutic range (Figure 4G).

Therapeutic management following the results of assays

Seventy six assays were performed in emergency departments, 28 were included in and 48 out the therapeutic ranges, the percentage of hospitalized patients were 53.6% (15/28) and 52.1% (25/48), respectively. No significant difference was found between patients with antiepileptic concentrations in and those outside therapeutic ranges regarding the rate of hospitalization (p=0.9) (Figure 5A).

Similarly, the dosage changes as well as the treatment changes were compared with the assay results for all prescribing services. Patients with plasma concentrations in the therapeutic ranges had more dosage changes (27.4% (34/124)) compared to those with plasma concentrations outside the therapeutic ranges (8.9% (7/78)) (p=0.0015). However, the background treatment change (discontinuation or addition of an antiepileptic drug) was not significantly different between these two groups (12.9% (16/124) and 7.7% (6/78) respectively) (p=0.247). The lack of change in antiepileptic treatment (dosage modification, addition or discontinuation of treatment) was therefore more frequent when plasma concentrations were outside therapeutic ranges (83.3% (65/78) compared to 59.7% (74/124) when plasma concentrations were in therapeutic ranges) (p=0.0004) (Figure 5B).

Discussion

Of the 242 assays performed since 2015, the vast majority (N=207) are in epileptic patients on whom the analyses have been targeted. Patients with lamotrigine therapy associated with levetiracetam were included in each group, so the patient number is lower than the assay number. From a general point of view, it is interesting to note that the most prescribing services of these assays are the emergency, intensive care unit, LTCU and neurology departments. These results seem consistent, given the reasons for the assay recommended by the HAS (2). In our study, 85.5% of the assays were in compliance with the 2007 HAS recommendations, indicating that clinicians generally complied with them well (Appendix A).

The results of lamotrigine and levetiracetam assays according to the reasons for prescription show a strong consistency with clinical intuition. The clearest examples concerns proven or suspected poor compliance, for which the highest proportion of underdose is found (Figure 4D). While measurement for suspected overdose found a high proportion of overdose (40%), we also found a high proportion of assays in therapeutic range. Measurement performed for therapeutic adjustment also found the highest proportion of assays in the therapeutic range (Figure 4A). However, data from the literature defined therapeutic range as the range of drug concentrations that provide best achievable response in a given patient [START_REF] Patsalos | Antiepileptic drugsbest practice guidelines for therapeutic drug monitoring: A position paper by the subcommission on therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies[END_REF]. Thus, a drug concentration considered in therapeutic standardized reference range can be considered underdose or overdose in some individuals, explaining our findings. Here, the use of 6 to 40 mg/L as levetiracetam therapeutic range can constitute an overstatement of assays in therapeutic range, where the use of 12-46 mg/L therapeutic range according to Jacob and Nair (2016) would have found more underdose [START_REF] Jacob | An Updated Overview on Therapeutic Drug Monitoring of Recent Antiepileptic Drugs[END_REF]. This limitation could explain why only 20% of assays performed in case of exacerbation or resurgence of epileptic seizures reported sub-therapeutic concentrations.

Considering hospitalization as a criterion for the severity of the disease, it seems consistent that an assay outside the therapeutic ranges should not be associated with a decision to hospitalize. Furthermore, these findings must be interpreted in the context of two potential limitations. Firstly, the retrospective design limited available data to those reported by physicians. This implies that unexplained discomfort reports could have been related to clinical signs of overdose and therefore associated with assays for suspicion of overdose. Considering that the medical records did not mention it, we classified these cases as non-recommended indications. Secondly, it is not excluded that (i) before receiving the result, another cause may be found, leading the patient to an emergency exit and (ii) that all assays were not known before patients were hospitalized.

The percentage of dosage change is significantly higher when plasma concentrations are in therapeutic ranges (p=0.0015). The lack of change in antiepileptic treatment (dosage modification, addition or discontinuation of treatment) was more frequent when plasma concentrations were outside therapeutic ranges (p=0.0004). These results suggest a re-evaluation of antiepileptic treatment mainly based on clinical criteria. In the literature, [START_REF] Kozer | Utility of antiepileptic drug monitoring in the pediatric emergency department[END_REF] conclude in a similar way, reporting that the antiepileptic drug assays outside a context of pharmacological therapeutic monitoring does not provide significant added value in practice and does not lead to a change in management in a paediatric emergency department [START_REF] Kozer | Utility of antiepileptic drug monitoring in the pediatric emergency department[END_REF]. As Selim and Cichowski (2018) point out, a dilemma arises regarding the management of the often expensive and unreliable assay result, which in some cases leads to unnecessary dosage changes that may increase the risk of adverse reactions [START_REF] Selim | Antiepileptic Drug Monitoring[END_REF]. In the case of persistent seizures, however, an assay may fall within the therapeutic range to assess the possibility of dosage increases. Here, we were limited by the lack of visibility on follow-up outside the hospital. It is therefore not excluded that treatment changes may have been made subsequently.

In the context of pharmacological therapeutic follow-up, the time of sampling, the patient's clinical condition, or the therapeutic ranges targeted by clinicians, are essential information for interpreting the result and therefore for customizing treatment [START_REF] Kang | Overview of Therapeutic Drug Monitoring[END_REF]. From an analytical point of view, good practices for the antiepileptic drugs determination recommend blood sampling just before the next oral dose, a condition in which the target concentrations have been defined [START_REF] Patsalos | Antiepileptic drugsbest practice guidelines for therapeutic drug monitoring: A position paper by the subcommission on therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies[END_REF]. In the case of analyses requested by the intensive care and emergency services, the hospital context is rarely compatible with this condition and samples are taken at all times. This parameter constitutes a major bias in a result report alone, due to the lack of visibility on the drug pharmacokinetics, and makes it difficult to interpret the result. Consequently, the interest of these assays is limited here to the comparison of the patient's clinical condition with a plasma concentration at the same time, and therefore does not constitute a pharmacological therapeutic follow-up [START_REF] Jacob | An Updated Overview on Therapeutic Drug Monitoring of Recent Antiepileptic Drugs[END_REF][START_REF] Kang | Overview of Therapeutic Drug Monitoring[END_REF][START_REF] Landmark | Dosing strategies for antiepileptic drugs: from a standard dose for all to individualised treatment by implementation of therapeutic drug monitoring[END_REF][START_REF] Patsalos | Therapeutic Drug Monitoring of Antiepileptic Drugs in Epilepsy: A 2018 Update[END_REF]. However, despite the absence of the time of sampling, lamotrigine and levetiracetam assays remain relevant for this study, considering (i) that according to the levetiracetam monography, the maximal concentration at the steady state for 1000 mg twice a day is 43 µg/mL, and thus nearly included in the therapeutic ranges and (ii) that lamotrigine elimination half-life is between 15h and 35h, showing a poor variation in plasma concentrations between two drug intakes when the steady state is reached.

This study highlights the difficulties encountered to obtain the information necessary for optimal care. Therefore, a relevant interpretation of the antiepileptic drug assays results in clinical practice remains difficult as it stands. These data reinforce the major interest of clinical-biological dialogue in optimizing individualized treatments and increasing its impact on medical care.

Conclusion

The requests analysis for antiepileptic drug assays at the University Hospital of Rennes reveals that clinicians are following HAS recommendations well. In addition, the assays results are mainly consistent with clinical intuition, suggesting a real added value in the patient management. However, the consequences on medical care seem limited. This assessment illustrates the importance of strengthening the dialogue between pharmacists, biologists and clinicians.

Conflict of interest

The authors declare no conflict of interest

Figure 1 :

 1 Figure 1 : Number of assays requests at the Laboratory of Biological and Forensic Toxicology at the University Hospital of Rennes for lamotrigine and levetiracetam determined by mass spectrometry (LC-MS/MS) from 2015 to 2018.

Figure 2 :

 2 Figure 2 : Number of antiepileptic drug assays performed according to their reasons: in accordance with HAS 2007 recommendations (therapeutic adjustment, specific clinical situation, suspicion of overdose, proven or suspected poor compliance, initiation of treatment, drug interaction management) and excluding HAS 2007 recommendations (routine monitoring, unexplained discomfort, and Other (follow-up of treatment after dosage modification or not, without seizure recurrence)).

Figure 3 :

 3 Figure 3 : Representation in a box and whisker plot of plasma concentrations (mg/L) of lamotrigine (A) and levetiracetam (B) in epileptic patients. The blue, green and red areas correspond respectively to underdose, results in the therapeutic range, and overdose.

Figure 4 :

 4 Figure 4 : Antiepileptic assay results expressed as a percentage of underdose, therapeutic range and overdose (levetiracetam and lamotrigine combined) according to the reasons for prescription: (A) Therapeutic adjustment in case of seizure recurrence or aggravation, (B) State of epilepsy, (C) Pregnancy, (D) Proven or suspected non-compliance, (E) Suspicion of overdose, (F) Initiation of treatment, (G) Assessment of unexplained discomfort.

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Figure 5 :

 5 Figure 5 : Medical management based on the results of lamotrigine and levetiracetam assays. (A) Percentage of hospitalization and return home following a visit to the emergencies. (B) Percentage of antiepileptic dosage change, treatment change (discontinuation of antiepileptic +/-introduction of a new antiepileptic treatment) and no change in treatment.

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