domain: sdv chim

text:
Patricia Leme Goto

Mathieu Cinato

Fadi Merachli

Bohdana Vons

Tony Jimenez

Dimitri Marsal

Nika Todua

Halina Loy

Yohan Santin

Stéphanie Cassel

Muriel Blanzat

Helene Tronchere

Christophe Dejugnat

Oksana Kunduzova

Frederic Boal
email: frederic.boal@inserm.fr

In vitro and in vivo cardioprotective and metabolic efficacy of vitamin E TPGS/Apelin

Keywords: cardiac hypertrophy, obesity, oxidative stress, surfactant

Aims: Apelin and vitamin E have been proposed as signaling molecules, but their synergistic role is unknown. The aim of this work was to develop vitamin E TPGS/Apelin system to test their cardioprotective and metabolic efficacy in vitro and in vivo. Methods: FDA-approved surfactant D-αtocopheryl polyethylene glycol 1000 succinate (TPGS-1000) and Apelin complex were characterized by physico-chemical methods (CMC determination, dynamic light scattering and circular dichroism).

In vitro studies were carried out on H9C2 cardiomyoblasts and isolated murine cardiomyocytes. In vivo studies were performed in isoproterenol-and high-fat diet-induced cardiac remodeling models in mice. Results: We found that vitamin E TPGS/Apelin provide cardioprotective and metabolic efficacy in vitro and in vivo. In vitro studies revealed that vitamin E TPGS/Apelin reduces hypoxiainduced mitochondrial ROS production in cultured cardiomyocytes and H9C2 cardiomyoblasts. In addition, vitamin E TPGS/Apelin confers apoptotic response to hypoxic stress in cells. In a mouse model of isoproterenol-induced cardiac injury, TPGS is not able to affect cardiac remodeling, however combination of vitamin E TPGS and Apelin counteracts myocardial apoptosis, oxidative stress, hypertrophy and fibrosis. Furthermore, combination treatment attenuated obesity-induced cardiometabolic and fibrotic remodeling in mice. Conclusion: Together, our data demonstrated the therapeutic benefits of vitamin E TPGS/Apelin complex to combat cardiovascular and metabolic disorders.

Introduction

The global prevalence of obesity and cardiovascular diseases has markedly increased over the past several decades in both developed and developing nations [START_REF] Aurigemma | Cardiac remodeling in obesity[END_REF]. Apelin is a highly conserved peptide from the adipokine family and a key regulator of cardiovascular and metabolic status [START_REF] Falcao-Pires | The apelinergic system: a promising therapeutic target[END_REF]. Apelin exerts strong inotropic actions [START_REF] Kleinz | Emerging roles of apelin in biology and medicine[END_REF] and regulates cardiac hypertrophy in response to pathological stress, activating the antioxidant enzyme catalase [START_REF] Foussal | Activation of catalase by apelin prevents oxidative stress-linked cardiac hypertrophy[END_REF]. Apelin was also shown to reduce cardiomyocyte cell death [START_REF] Boal | Apelin regulates FoxO3 translocation to mediate cardioprotective responses to myocardial injury and obesity[END_REF][START_REF] Boal | Apelin-13 administration protects against ischaemia/reperfusion-mediated apoptosis through the FoxO1 pathway in high-fat diet-induced obesity[END_REF] and fibrotic remodeling of the heart [START_REF] Pchejetski | Apelin prevents cardiac fibroblast activation and collagen production through inhibition of sphingosine kinase 1[END_REF]. Moreover, it has been reported by us and others that apelin improves diabetic cardiomyopathy phenotype [START_REF] Alfarano | Transition from metabolic adaptation to maladaptation of the heart in obesity: role of apelin[END_REF][START_REF] Zeng | Apelin gene therapy increases myocardial vascular density and ameliorates diabetic cardiomyopathy via upregulation of sirtuin 3[END_REF][START_REF] Ceylan-Isik | Apelin administration ameliorates high fat diet-induced cardiac hypertrophy and contractile dysfunction[END_REF], promotes survival of transplanted mesenchymal cells [START_REF] Liang | Therapeutic efficacy of apelin on transplanted mesenchymal stem cells in hindlimb ischemic mice via regulation of autophagy[END_REF] and that apelin plasma levels are increased in obese patients after myocardial infarction [START_REF] Boal | Apelin regulates FoxO3 translocation to mediate cardioprotective responses to myocardial injury and obesity[END_REF], opening new therapeutic avenues for the treatment of cardiovascular and metabolic disorders.

Vitamin E is a fat-soluble antioxidant vitamin whose deficiency results from several chronic diseases associated with fat malabsorption, such as cystic fibrosis, Crohn's disease, chronic cholestasis, pancreatic insufficiency or gastric bypass in obese patients [START_REF] Azzi | Many tocopherols, one vitamin E[END_REF]. Antioxidant vitamins are commonly prescribed to patients after cardiac transplant, in order to limit oxidative stress and therefore transplant rejection [START_REF] Fang | Effect of vitamins C and E on progression of transplant associated arteriosclerosis: a randomised trial[END_REF]. Nevertheless, the clinical efficacy of vitamin E supplementation is not fully established [START_REF] Jaxa-Chamiec | Effects of vitamins C and E on the outcome after acute myocardial infarction in diabetics: A retrospective, hypothesis-generating analysis from the MIVIT Study[END_REF][START_REF]MRC/BHF Heart Protection Study of antioxidant vitamin supplementation in 20,536 high-risk individuals: a randomised placebo-controlled trial[END_REF][START_REF] Yusuf | Vitamin E supplementation and cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators[END_REF]. In recent years, interest has grown on D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS-1000), a water-soluble derivative of natural vitamin E. TPGS-1000 is formed by the esterification of D-α-tocopheryl succinate with polyethylene glycol (PEG), resulting in a hydrophilic polar PEG head and a lipophilic tocopheryl tail, providing TPGS-1000 with the amphipathic properties of surfactants [START_REF] Sadoqi | Investigation of the micellar properties of the tocopheryl polyethylene glycol succinate surfactants TPGS 400 and TPGS 1000 by steady state fluorometry[END_REF]. TPGS-1000 is a GRAS (generally regarded as safe)-listed supplement and is FDAapproved as a drug solubilizer. Recently, it has been shown to be effective in several anti-cancer drug delivery systems, including doxorubicin [START_REF] Cao | Doxorubicin conjugated to d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS): Conjugation chemistry, characterization, in vitro and in vivo evaluation[END_REF] and curcumin [START_REF] Rachmawati | Multiple Functions of D-α-Tocopherol Polyethylene Glycol 1000 Succinate (TPGS) as Curcumin Nanoparticle Stabilizer: In Vivo Kinetic Profile and Anti-Ulcerative Colitis Analysis in Animal Model[END_REF], improving their therapeutic value. Moreover, it is widely used as a water-soluble vitamin E supplement for people with impaired uptake or chronic liver diseases, in particular in children with chronic cholestasis [START_REF] Sokol | Multicenter trial of dalpha-tocopheryl polyethylene glycol 1000 succinate for treatment of vitamin E deficiency in children with chronic cholestasis[END_REF] and short bowel syndrome [START_REF] Traber | Efficacy of water-soluble vitamin E in the treatment of vitamin E malabsorption in short-bowel syndrome[END_REF]. These disorders may affect cardiac function and predispose to the development of cardiomyopathy [START_REF] Møller | Cirrhotic cardiomyopathy: a pathophysiological review of circulatory dysfunction in liver disease[END_REF][START_REF] Fouad | Hepato-cardiac disorders[END_REF]. More recently, the effect of TPGS-1000 as a vitamin E alternative has been investigated in a rat model of cardiac transplant [START_REF] Nguyen | Beneficial effect of alphatocopheryl succinate in rat cardiac transplants[END_REF], opening new perspectives on the use of its surfactant properties in the field of cardiovascular diseases. In this context, combination of TPGS-S 1000 and apelin is a promising approach to combat complex or multifactorial disorders including S¡ obesity and heart failure. However, To to d¢£¤¥ £¦¤ §¤ ¨© study on the interactions of TPGS-1000 S and apelin. In this study we characterized TPGS-1000/Apelin complex to test their cardioprotective benefits in vitro and in vivo. We found that TPGS-1000/Apelin complex counteracts adverse cardiometabolic and fibrotic remodeling in mice. In addition, in vitro studies revealed that TPGS-1000/Apelin complex confers protection against hypoxia-induced cell death and oxidative stress.

Materials and Methods

Reagents

[Pyr 1 ]-Apelin 13 (stated as apelin in the text) was purchased from Genecust Europe. D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS-1000), isoproterenol hypochloride and Hoechst 34580 were obtained from Sigma-Aldrich (France).

Cell culture and treatments

The H9C2 rat embryonic cardiomyoblastic cell line was cultured in DMEM medium (Life technologies) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Life technologies) in a 37°C, 5% CO2 incubator. Primary cardiomyocytes were isolated from adult C57BL6/J mice hearts as previously described [START_REF] Fazal | Multifunctional Mitochondrial Epac1 Controls Myocardial Cell Death[END_REF]. Cell viability was assessed using MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide) according to manufacturer's instructions (Sigma). For hypoxic treatment, cells were pretreated for 30 min with apelin 10 -7 M as described [START_REF] Boal | Apelin regulates FoxO3 translocation to mediate cardioprotective responses to myocardial injury and obesity[END_REF], TPGS-1000 or apelin+TPGS-1000 and subjected to normoxia (5% CO2, 21% O2, balance N2) or hypoxia in a hypoxic chamber (5% CO2, 1% O2, balance N2) for 2 hours for ROS production measurement and for 16 hours followed by a reoxygenation period of 4 hours for apoptosis evaluation. Quantification was performed as described [START_REF] Tronchere | Inhibition of PIKfyve prevents myocardial apoptosis and hypertrophy through activation of SIRT3 in obese mice[END_REF].

Evaluation of apoptosis and ROS production

H9C2 apoptosis was assessed by nuclear staining with 5µg/ml Hoechst 34580 for 20min. Cells were then live-imaged on a wide-field Leica microscope. Pyknotic nuclei were counted and expressed as percentage of total nuclei. Apoptosis level on heart cryosections and cells was assessed using DeadEnd Fluorometric TUNEL system according to manufacturer's instructions (Promega).

Mitochondrial superoxide levels on heart cryosections and cells were assessed with MitoSOX Red fluorescent probe (Life Technologies) and imaged by confocal microscopy as described [START_REF] Tronchere | Inhibition of PIKfyve prevents myocardial apoptosis and hypertrophy through activation of SIRT3 in obese mice[END_REF].

Cell lysate and Western-blotting

Proteins from H9C2 cells were extracted using RIPA buffer supplemented with proteases inhibitors and western-blots were performed as described before [START_REF] Boal | Apelin regulates FoxO3 translocation to mediate cardioprotective responses to myocardial injury and obesity[END_REF]. Antibodies used are as follows: cleaved-Caspase 3 from Cell Signaling Technology and RhoGDI from Santa Cruz.

Animal studies

The investigations conform to the Animal Care and Use Committee of the University of Toulouse, 1985). For isoproterenol induced cardiac hypertrophy, two-month-old C57BL6/J mice were pretreated with one intraperitoneal injection of vehicle only (PBS), apelin (100µg/kg/d), TPGS-1000 (1.32µmol/kg/d) or the combinatory therapy. Apelin amount used here was as described before [START_REF] Boal | Apelin regulates FoxO3 translocation to mediate cardioprotective responses to myocardial injury and obesity[END_REF] and the TPGS amount was set to maintain the Apelin:TPGS 1/20 ratio as for the in vitro studies and physico-chemical characterizations. Mice were then treated with isoproterenol (i.p., 15mg/kg/d) in the continuous presence of apelin, TPGS-1000, the combinatory therapy or vehicle only for 8 consecutive days. For obesity-linked cardiomyopathy, two-month old C57BL6/J mice were fed with high fat diet (HFD, 45% fat, Research Diet D12451) for 14 months. HFD-fed mice were implanted with AZELT osmotic minipumps to deliver vehicle only (PBS) or the combinatory therapy apelin+TPGS-1000 for 28 days. Glucose tolerance test was performed on fasted mice by intraperitoneal injection of glucose (1g/kg). Blood glucose levels from tail vein were monitored over time using a glucometer (Accu-check, Roche Diagnostics). Echocardiographic studies were performed as described [START_REF] Tronchere | Inhibition of PIKfyve prevents myocardial apoptosis and hypertrophy through activation of SIRT3 in obese mice[END_REF].

Quantitative RT-PCR analysis

Total RNAs were extracted from mice heart in Qiazol using FastPrep lysing matrix M tubes (MP Biomedicals) and further purified using RNAeasy mini kit (Qiagen). Total RNAs were reverse transcribed using superscript II reverse transcriptase (Invitrogen). Real time quantitative PCR was performed as previously described (Alfarano et al., 2014) on a StepOnePlus realtime PCR system (Thermo Fischer scientific). Primers used on this study are as follows: collagen I 5'-TGTGTGCGATGACGTGCAAT-3' (forward) and 5'-GGGTCCCTCGACTCCTACA-3' (reverse); collagen III 5'-AAGGCGAATTCAAGGCTGAA-3 (forward) and 5'-TGTGTTTAGTACAGCCATCCTCTAGA-3' (reverse) and alpha-SMA 5'-GTCCCAGACATCAGGGAGTAA-3' (forward) and 5'-TCGGATACTTCAGCGTCAGGA-3' (reverse); PGC1-b 5'-TTGAGGTGTTCGGTGAGATTGTAG-3' (forward) and 5'-GAAGGTGATAAAACCGTGCTTCTG-3' (reverse); ANP 5'-AGAGTGGGCAGAGACAGCAAA-3' (forward) and 5'-AAGGCCAAGACGAGGAAGAAG-3' (reverse); BNP 5'-GCACAAGATAGACCGGATCG-3' (forward) and 5'-CCCAGGCAGAGTCAGAAAC-3' (reverse).

Morphology

Hematoxylin-eosin (H/E) staining of heart cryosections was performed using standard method.

Myocytes hypertrophy was assessed by measuring cardiomyocyte cross sectional area on either H/E or on fluorescent WGA stained cryosections. Cardiac fibrosis was assessed using sirius red staining according to standard method or by fluorescent WGA staining according to [START_REF] Emde | Wheat germ agglutinin staining as a suitable method for detection and quantification of fibrosis in cardiac tissue after myocardial infarction[END_REF].

Statistical analysis

Data are expressed as mean+/-SEM. Comparison between two groups was performed by Student's two-tailed t-test while comparison of multiple groups was performed by one-way ANOVA followed by a Bonferroni's post hoc test using GraphPad Prism version 5.00 (GraphPad Software, Inc).

Results

Cumulative protection of vitamin E TPGS/Apelin against mitochondrial oxidative stress

We first investigated the dose-dependent effects of TPGS-1000 on H9C2 cell viability. As shown in Fig. 1a, treatment with up to 2µM TPGS-1000 for 24h did not alter cell viability. However, higher concentration decreased dramatically H9C2 cell viability, down to 25% for 20µM treatment.

Therefore, we used TPGS-1000 on H9C2 cells at 2µM final concentration throughout this study. In order to evaluate the antioxidant properties of TPGS-1000, apelin and vitamin E TPGS/Apelin complex, we measured mitochondrial O2 -production by MitoSOX Red staining of living H9C2 cells exposed to hypoxia. As shown in Fig. 1b, while hypoxia treatment induced an excessive production of mitochondrial ROS, treatment of cells with TPGS-1000 alone (Fig. 1b, HT) or apelin alone (Fig. 1b, HA) totally abrogated mitochondrial O2 -production induced by hypoxia. Interestingly, quantification indicates a cumulative effect of vitamin E TPGS/Apelin (Fig. 1b, HTA). As undifferentiated H9C2 cardiomyoblasts may not represent cardiomyocytes, we investigated the effect of TPGS-1000 and apelin on mitochondrial O2 -production in primary cardiomyocytes isolated from adult mice. As shown in Fig. 1c, both apelin and TPGS-1000 strongly reduced mitochondrial O2 -production induced by hypoxia. Similarly to what we observed in H9C2 cells, the combinatory therapy showed a cumulative anti-oxidant effect of TPGS1000 and apelin when added together (Fig. 1c).

As TPGS-1000 is a well-known surfactant commonly used to formulate molecules, we investigated the physico-chemical properties of TPGS-1000 in the presence of apelin. Surface tension measurements showed that the critical micelle concentration (CMC) of TPGS-1000 was affected by the presence of apelin, decreasing from 10µM to 4µM (supplementary Fig. 1a). This suggests that apelin slightly promotes the micellization of TPGS-1000. Moreover, some signs of a pre-aggregation could be detected at very low concentrations, confirming this hypothesis. In contrast, dynamic light scattering (DLS) measurements showed that apelin had no effect on the mean hydrodynamic radius of TPGS-1000 micelles, remaining at 8nm (supplementary Fig. 1b). Moreover, using circular dichroism spectroscopy we found that TPGS-1000 did not affect structuration of apelin: even if the profile was slightly affected by the presence of the surfactant, it remained typical of a coil conformation (supplementary Fig. 1c). Altogether these results indicate that, even if the two compounds might interact weakly in the mixture, the main properties of TPGS-1000 and apelin remained conserved without loss of activity.

Vitamin E TPGS/Apelin counteracts hypoxia-induced apoptotic cell death in H9C2 cells

Excessive ROS production is linked to mitochondrial damage, activation of apoptotic cascades and ultimately cell death [START_REF] Tsutsui | Oxidative stress and heart failure[END_REF]. We next evaluated the effects of Vitamin E TPGS/Apelin complex on hypoxia-induced apoptosis in H9C2 cardiomyoblasts. Apoptotic cell death was assessed either by monitoring pyknotic nuclei (Fig. 2a andc) and by TUNEL staining (Fig. 2b andd). Whereas apelin was potent to prevent hypoxia-induced apoptotic cell death, TPGS-1000 had no effect. Importantly, treatment of cells with Vitamin E TPGS/Apelin complex significantly reduced the number of apoptotic cells in H9C2 cells under hypoxia (Fig. 2a-e). This was further confirmed by monitoring the cleavage of caspase 3, a bona fide marker of apoptotic cascade activation (Fig. 2e andf). This suggests that Vitamin E TPGS/Apelin provide protection against apoptosis under stress conditions.

To further investigate how TPGS-1000/apelin reduced ROS production and apoptosis, we quantified the expression level of mitochondrial biogenesis regulatory factor PGC1-b. As shown in Fig. 2g, we found that long term hypoxic challenge dramatically reduced PGC1-b expression. Importantly, apelin, but not TPGS-1000, restored PGC1-b level (Fig. 2g), suggesting that TPGS-1000, unlike apelin, is not able to induce mitochondrial biogenesis.

Effect of Vitamin E TPGS/Apelin on isoproterenol-induced cardiac injury in vivo

In order to investigate the effect of Vitamin E TPGS/Apelin in vivo, we resorted to a chemicallyinduced cardiac remodeling mouse model. Isoproterenol (ISO) is a beta-adrenergic agonist commonly used in vivo to induce cardiac hypertrophy, ROS production and apoptosis [START_REF] Tronchere | Inhibition of PIKfyve prevents myocardial apoptosis and hypertrophy through activation of SIRT3 in obese mice[END_REF]. As shown by hematoxylin-eosin staining, chronic treatment with ISO induced cardiac hypertrophy (Fig. 3a,H/E).

These results were confirmed using the fluorescent probe wheat-germ agglutinin (Fig. 3a, WGA) and quantification of cross-sectional cardiomyocyte area (Fig. 3b). Strikingly, treatment with Vitamin E TPGS/Apelin complex or apelin attenuated ISO-induced cardiac hypertrophic response, while TPGS-1000 treatment had no significant effects (Fig. 3). Interestingly, TPGS-1000 did not alter apelin antihypertrophic properties (Fig. 3, ISO+TPGS+A). This was further confirmed by measuring the expression levels of the hypertrophic markers ANP and BNP (Fig. 3c andd). Next we explored the antioxidant properties of Vitamin E TPGS/Apelin complex versus apelin and TPGS-1000 in vivo. As shown in Fig. 4a, both TPGS/Apelin complex and apelin markedly decreased ISO-induced myocardial mitochondrial O2 -production in cardiac tissue, while TPGS-1000 had no effect. To examine the antiapoptotic activity of TPGS/Apelin complex versus TPGS-1000 and apelin in vivo, we resorted to the TUNEL-staining of apoptotic cells on heart cryosections. As shown in Fig. 4b, ISO-treatment induced an increase in myocardial apoptotic cell death, which was prevented in vitamin E TPGS/Apelintreated or apelin-treated mice. TPGS-1000 treatment did not affect ISO-induced apoptotic cell death in cardiac tissue in treated animals. Together, these data revealed anti-apoptotic activity of vitamin E TPGS/Apelin in cardiac remodeling in vivo.

Excessive ROS production and massive loss of cardiomyocytes by apoptosis lead to the development of cardiac fibrosis [START_REF] González | Myocardial Interstitial Fibrosis in Heart Failure[END_REF]. Therefore we examined the effects of vitamin E TPGS/Apelin on cardiac study we characterized vitamin E TPGS/Apelin complex to evaluate their cardioprotective benefits in vitro and in vivo. We characterized the cardiometabolic efficacy of vitamin E TPGS/Apelin in the progressive myocardial remodeling processes. In a mouse model of isoproterenol-induced cardiac injury, vitamin E TPGS/Apelin counteracts myocardial apoptosis, oxidative stress, hypertrophy and fibrosis. Furthermore, we provide the first evidence that vitamin E TPGS/Apelin attenuates obesityinduced cardiometabolic and fibrotic remodeling in mice. In vitro studies reveal vitamin E TPGS/Apelin-dependent antioxidant and anti-apoptotic reprogramming in cells under hypoxic stress.

We found that both TPGS-1000 and apelin protect cultured cardiomyoblasts and primary cardiomyocytes against oxidative stress in hypoxic challenge in a cumulative manner. However, we show that TPGS-1000 is ineffective to prevent apoptotic cell death as compared to apelin treatment.

This discrepancy between TPGS-1000 and apelin suggests that they may use separate pathways/mechanisms to exert their antioxidant properties. Indeed, we and others previously demonstrated that apelin, through the binding of its receptor AJP, 1/ triggers mitochondrial biogenesis through activation of the transcription factors TFAM and NRF1 [START_REF] Alfarano | Transition from metabolic adaptation to maladaptation of the heart in obesity: role of apelin[END_REF], 2/ activates the antioxidant enzyme catalase [START_REF] Foussal | Activation of catalase by apelin prevents oxidative stress-linked cardiac hypertrophy[END_REF], 3/ regulates the FoxO family of transcription factors [START_REF] Boal | Apelin regulates FoxO3 translocation to mediate cardioprotective responses to myocardial injury and obesity[END_REF][START_REF] Boal | Apelin-13 administration protects against ischaemia/reperfusion-mediated apoptosis through the FoxO1 pathway in high-fat diet-induced obesity[END_REF] and 4/ activates the AMPK pathway [START_REF] Dray | Apelin stimulates glucose utilization in normal and obese insulin-resistant mice[END_REF]. On the other hand, TPGS-1000 is known to act through the intracellular liberation of the vitamin E moiety, giving direct antioxidant action in cells, as previously demonstrated in a rat cardiac transplant model [START_REF] Nguyen | Beneficial effect of alphatocopheryl succinate in rat cardiac transplants[END_REF]. Consistently, we found that TPGS-1000 is not able to restore the expression of the mitochondrial biogenesis regulatory factor PGC1-b after longterm hypoxic challenge. This could explain the lack of effect of TPGS-1000 in long-term challenges, both in vitro and in vivo.

In vivo, we found that vitamin E TPGS/Apelin counteracts cardiac hypertrophy, oxidative stress and cardiomyocyte cell death induced by isoproterenol, culminating in reducing fibrotic remodeling of the heart. However, we demonstrated that TPGS-1000 alone did not provide protection against myocardial hypertrophy, oxidative stress, apoptosis and activation of fibrotic reprogramming of the myocardium. One might hypothesize that a poor bioavailability of TPGS-1000 in cardiac tissue could explain this lack of effect. Although TPGS-1000 is well tolerated in vivo and has a good bioavailability, previous studies suggested that it has a weak tropism for cardiac tissue [START_REF] Tesan | Radiolabeling and biological characterization of TPGS-based nanomicelles by means of small animal imaging[END_REF]. Our physico-chemical characterization shows that apelin slightly decrease the CMC of TPGS-1000 in solution, from 10µM to 4µM, not reaching the non-toxic concentration of 2µM. Nevertheless, we found here that chronic treatment with TPGS-1000 in mice failed to protect the heart against ISO-induced cardiac remodeling. Moreover, this is consistent with the fact that TPGS-1000 did protect neither cardiomyoblasts nor cardiomyocytes against H/R-induced apoptotic cell death in long term challenge.

TPGS-1000 is widely used for its surfactant properties, namely as a solubilizer, emulsifier and bioavailability enhancer to increase drug delivery [START_REF] Yang | Recent Advances in the Application of Vitamin E TPGS for Drug Delivery[END_REF]. The initial aim of this study was to combine apelin with TPGS-1000 in order to improve apelin cardioprotective effects. Our physico-chemical characterization shows that apelin slightly modify the CMC of TPGS-1000 in solution, from 10µM to 4µM. Although apelin clearly decreases TPGS-1000 CMC, it does not reach the non-toxic concentration of 2µM in vitro. More work needs to be done in order to decrease TPGS-1000 CMC in a concentration range compatible with cardiomyoblasts viability. Moreover, a recent study confirmed the cytotoxicity of TPGS-1000, showing that it induces cell cycle arrest and even apoptosis on breast cancer cells [START_REF] Neophytou | d-alpha-tocopheryl @A/19 polyethylene glycol succinate (TPGS) induces cell cycle arrest and apoptosis selectively in Survivin-overexpressing breast cancer cells[END_REF], and thus care must be taken for its use in drug delivery.

Furthermore, in a mouse model of obesity-induced cardiomyopathy, we found that vitamin E TPGS/Apelin counteracts obesity-induced myocardial and metabolic abnormalities. Specifically, chronic treatment with TPGS/Apelin attenuated HFD-mediated cardiomyocyte death and cardiac dysfunction. In addition, we found that vitamin E TPGS/Apelin improved glucose tolerance in HFD-fed mice, suggesting that TPGS/Apelin modulates pathological remodeling of the diabetic heart in obese conditions. The development of treatments involving combinations of drugs is a promising approach ! to combat complex or multifactorial disorders including obesity and heart failure. This study provides " #$/19 the first evidence for the therapeutic benefits of vitamin E TPGS/Apelin to prevent cardiovascular and %&& metabolic complications in obesity-linked heart failure. However, a limitation of this study is the %&' absence of the apelin-treated HFD-fed mice. Nevertheless, we previously reported apelin-mediated %&( cardioprotective responses to myocardial injury in HFD-induced obesity )0&1. In isoproterenol-%&2 treated mice, apelin and vitamin E TPGS/Apelin complex have similar results, suggesting that the %'3 addition of vitamin E TPGS exerts no further benefit. The potential benefit of vitamin E TPGS/Apelin %'4 complex need to be further addressed in different experimental models of heart failure, such as %'% transverse aortic constriction or ischemia-reperfusion. %'5

In summary, our findings demonstrated the protective effects of vitamin E TPGS/Apelin on adverse 274 tissue reprogramming of the myocardium and metabolic defects in physiopathological context. 275 Moreover, we revealed that oxidative stress and apoptotic cell death, key components in the 276 development of cardiac injury, are attenuated by vitamin E TPGS/Apelin in both isoproterenol-and 277 HDF-induced myocardial remodeling. These findings may lead to novel approaches for the treatment 278 of adverse cardiovascular and metabolic complications linked to heart failure. 279

  INSERM/ENVT and to the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Council of Europe No 123, Strasbourg

Fig. 1 :

 1 Fig. 1: Vitamin E TPGS/Apelin attenuates mitochondrial ROS production in H9C2 cells and

Fig. 2 :

 2 Fig. 2: Vitamin E TPGS/Apelin prevents apoptotic responses to hypoxic stress. a. H92C cells were

Fig. 3 :

 3 Fig.3: Vitamin E TPGS/Apelin inhibits ISO-induced hypertrophic remodeling in vivo. a. Heart

Acknowledgements 280

We thank the Cellular Imaging Facility Rangueil-I2MC-TRI. This work was funded by Région Occitanie 281 (RBIO 14054336), INSERM, CNRS and Université Toulouse III. 282

fibrosis using Sirius red and fluorescent WGA staining in cardiac tissue from ISO-induced myocardial injury. As shown in Fig. 5, treatment with ISO induced the development of a pronounced perivascular and interstitial fibrosis in mice. While vitamin E TPGS/Apelin complex or apelin was able to efficiently reduce cardiac fibrosis, TPGS-1000 had no anti-fibrotic activity in ISO-remodeled hearts (Fig. 5).

Effect of vitamin E TPGS/Apelin on a HFD-induced diabetic cardiomyopathy

We next examined the effects of vitamin E TPGS/Apelin on myocardial and metabolic remodeling in mice exposed to HFD for 14 months. HFD-feeding resulted in the development of glucose intolerance, insulin resistance (Fig. 6a andb) and increased body weight (Fig. 6c). Echocardiographic analysis revealed left ventricular dysfunction as shown by the reduced ejection fraction (Fig. 6d) and fractional shortening (Fig. 6e). Treatment with vitamin E TPGS/Apelin improved glucose tolerance in HFD-fed mice (Fig. 6a andb). The anti-glycemic activity of the vitamin E TPGS/Apelin was associated with a reduction in body weight, although it did not reach statistical significance (Fig. 6c).

Importantly, vitamin E TPGS/Apelin treatment preserved cardiac function in obese mice as shown by the increase in EF and FS (Fig. 6d ande). This was accompanied by a reduction of cardiac hypertrophy as shown by cardiomyocytes cross-sectional area measurements (Fig. 7a), a decrease in myocardial mitochondrial O2 -production (Fig. 7b) and apoptosis (Fig. 7c). Interestingly, the combinatory therapy is potent to reduce myocardial fibrosis in HFD-fed mice (Fig. 8a). These results were confirmed by qPCR analysis of the expression of the myocardial pro-fibrotic markers aSMA, collagen I and collagen III (Fig. 8b).

Taken together, our results show for the first time that vitamin E TPGS/Apelin behaves as a competitive cardioprotective and metabolic modulator of myocardial remodeling processes.

Discussion

Apelin and vitamin E TPGS have been largely recognized for their antioxidant properties [START_REF] Foussal | Activation of catalase by apelin prevents oxidative stress-linked cardiac hypertrophy[END_REF][START_REF] Sadoqi | Investigation of the micellar properties of the tocopheryl polyethylene glycol succinate surfactants TPGS 400 and TPGS 1000 by steady state fluorometry[END_REF]. However, to date, there is no study regarding the interactions of vitamin E TPGS and apelin. In this

Disclosures 283

The authors have no conflicts of interest to declare. 284

Supplementary material 285

Supplementary methods and figures are provided separately. compared with ISO-treated mice.


Simon J Cleary

Carl Hobbs

Richard T Amison

Stephanie Arnold

Blaze G O'shaughnessy

Emma Lefrançais

Beñat Mallavia

Mark R Looney

Clive P Page

Dr Simon C Pitchford
email: simon.pitchford@kcl.ac.uk

LPS-induced lung platelet recruitment occurs independently from neutrophils, PSGL-1, and P-selectin

Keywords: Neutrophil-independent lung platelet recruitment Platelets, neutrophils, recruitment, lungs, LPS Platelets, neutrophils, recruitment, lungs, LPS

Platelets are recruited to inflammatory foci and contribute to host defence and inflammatory responses. Compared to platelet recruitment in haemostasis and thrombosis, the mechanisms of platelet recruitment in inflammation and host defence are poorly understood. Neutrophil recruitment to lung airspaces following inhalation of bacterial LPS requires platelets and PSGL-1 in mice. Given this association between platelets and neutrophils, we investigated whether recruitment of platelets to lungs of mice following LPS inhalation was dependent on PSGL-1, P-selectin, or interaction with neutrophils.

BALB/c mice were administered intranasal LPS (O55:B5, 5 mg/kg) and 48 hours later lungs were collected, and platelets and neutrophils were quantified in tissue sections by immunohistochemistry.

The effects of functional blocking antibody treatments targeting the platelet-neutrophil adhesion molecules P-selectin, or PSGL-1, or treatment with a neutrophil depleting antibody targeting Ly6G, were tested on the extent of LPS-induced lung platelet recruitment. Separately in Pf4-Cre×mTmG mice, two-photon intravital microscopy was used to image platelet adhesion in live lungs.

Inhalation of LPS caused both platelet and neutrophil recruitment to the lung vasculature. However, decreasing lung neutrophil recruitment by blocking PSGL-1, P-selectin, or depleting blood neutrophils had no effect on lung platelet recruitment. Lung intravital imaging revealed increased adhesion of platelets in the lung microvasculature which was not associated with thrombus formation.

In conclusion, platelet recruitment to lungs in response to LPS occurs through mechanisms distinct from those mediating neutrophil recruitment, or the occurrence of pulmonary emboli.

Introduction

Platelet and neutrophil recruitment to lungs are critical to host defence, but when excessive or inappropriate can also contribute to tissue damage in a range of inflammatory diseases including: acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and respiratory infections. 1,2 Research suggests that platelets can exert a controlling influence on neutrophil recruitment, particularly in acute inflammation in the lung, where responses are dependent upon the presence of platelets displaying adhesion molecules that interact with counterligands expressed on neutrophils, [START_REF] Kornerup | Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration[END_REF][START_REF] Page | Neutrophil and platelet complexes and their relevance to neutrophil recruitment and activation[END_REF][START_REF] Hidalgo | Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury[END_REF] signalling in platelets through the purinergic receptors P2Y 1 and P2Y 14 , [START_REF] Amison | RhoA signaling through platelet P2Y₁ receptor controls leukocyte recruitment in allergic mice[END_REF][START_REF] Amison | Lipopolysaccharide (LPS) induced pulmonary neutrophil recruitment and platelet activation is mediated via the P2Y1 and P2Y14 receptors in mice[END_REF] P-Rex1 and Vav guanine nucleotide exchange factors, [START_REF] Pan | P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of in fl ammation[END_REF] and the release of CCL5, CXCL4 and CXCL7 chemokines, [START_REF] Grommes | Disruption of platelet-derived chemokine heteromers prevents neutrophil extravasation in acute lung injury[END_REF] and serotonin, [START_REF] Duerschmied | Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice[END_REF] from platelets. These data provide evidence of plateletdependent signalling mechanisms by which platelets influence neutrophil recruitment. Associations between platelets and neutrophils are further observed in acute lung inflammation by the presence of heightened circulating blood platelet-neutrophil complexes, [START_REF] Ortiz-Muñoz | Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[END_REF] and platelet dependence of both neutrophil rolling, intravascular crawling and early diapedesis. [START_REF] Wang | P-selectin primes leukocyte integrin activation during inflammation[END_REF] Furthermore, platelets can sometimes be found associated with extravascular neutrophils in inflamed lungs. [START_REF] Ortiz-Muñoz | Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[END_REF][START_REF] Amison | Platelet Depletion Impairs Host Defence to Pulmonary Infection with Pseudomonas aeruginosa in Mice[END_REF][START_REF] Sreeramkumar | Neutrophils scan for activated platelets to initiate inflammation[END_REF][START_REF] Zuchtriegel | Platelets Guide Leukocytes to Their Sites of Extravasation[END_REF] How leukocytes are recruited from blood to tissues to act as major effectors of host defence and inflammation has become increasingly well characterised. 2,[START_REF] Ley | Getting to the site of inflammation: the leukocyte adhesion cascade updated[END_REF] The involvement of platelets in leukocyte recruitment in inflammation therefore adds a further point of control for coordinating inflammatory responses. 1,[START_REF] Semple | Platelets and the immune continuum[END_REF] Blood platelet recruitment to lesions in blood vessel walls is also becoming increasingly understood at a molecular level in the context of haemostasis and thrombosis. Similarly to leukocytes, platelets can independently respond to pathogen-associated molecular patterns, and importantly can be recruited from blood to accumulate in tissues following infection or different types of inflammatory insult to mediate temporally and spatially restricted signalling. 1 This platelet recruitment in host defence and inflammation can occur in the absence of any overt vascular trauma or thrombus formation through mechanisms that are incompletely understood, but which are distinct from those required for haemostasis and thrombosis. 1,[START_REF] Page | The involvement of platelets in non-thrombotic processes[END_REF] Intriguingly, given the involvement of platelets in neutrophil recruitment into lung airspaces, platelet recruitment to lungs following inflammatory responses originating elsewhere in the body, such as LPSinduced peritonitis, [START_REF] Andonegui | Platelets express functional Toll-like receptor-4[END_REF] or in the bloodstream following transfusion with anti-MHCI antibodies, [START_REF] Looney | Platelet depletion and aspirin treatment protect mice in a two-event model of transfusion-related acute lung injury[END_REF] has shown evidence of neutrophil dependence. Neutrophils have been identified as effector cells in mediating the responses to inhaled LPS, [START_REF] Grommes | Disruption of platelet-derived chemokine heteromers prevents neutrophil extravasation in acute lung injury[END_REF][START_REF] Savov | Neutrophils play a critical role in development of LPS-induced airway disease[END_REF] but the requirements for platelets in this context has not yet been explored. Understanding responses such as lung neutrophil and platelet recruitment, and how these are regulated may provide important information leading to new targets for the development of novel anti-inflammatory drugs.

The platelet adhesion molecules involved in the recruitment of platelets to lungs in host defence and inflammation have not yet been fully characterized. Potential candidates for this role are P-selectin, PSGL-1, a counter ligand of P-, E-and L-selectins, and ESL-1, a counter ligand to E-selectin. [START_REF] Kornerup | Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration[END_REF][START_REF] Hidalgo | Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury[END_REF][START_REF] Pan | P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of in fl ammation[END_REF][START_REF] Carlow | PSGL-1 function in immunity and steady state homeostasis[END_REF][START_REF] Frenette | P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo[END_REF][START_REF] Pitchford | Platelet P-selectin is required for pulmonary eosinophil and lymphocyte recruitment in a murine model of allergic inflammation[END_REF] Study of these adhesion molecules offers potential identification of similarities and differences between platelet and neutrophil recruitment in inflammation.

We therefore developed methods for imaging lung platelet recruitment following LPS inhalation in mice, and then investigated the effects of blockade of P-selectin or PSGL-1, or the selective depletion of neutrophils, on this response.

Materials and Methods

Readers are directed to the online supplementary material for methodology related to: collection of mouse lungs for histology, immunohistochemistry using fluorescent reporters, immunohistochemistry using bright field DAB reporter, analysis of images of lung sections, flow cytometry analysis, experiments involving dabigatran etexilate, leukocyte and platelet counts from blood microsamples, and bronchoalveolar lavage (BAL) collection.

Mice

Mice (BALB/c strain, Female, 6-12 weeks of age) used at King's College London were obtained from Charles River (Harlow, UK) and housed in non-barrier facilities. Animal experiments were conducted according to ARRIVE guidelines, and the Animals (Scientific Procedures) Act, 1986, and 2012 amendments following local ethical approval. Terminal anaesthesia was achieved using urethane (3 g/kg, i.p.). Weight changes were monitored as an index of general health status (Supplementary Figure 1B), and it was noted that intervention studies outlined below (e.g blockade of PSGL-1, and Pselectin; and neutrophil depletion) had no effect on this parameter (Supplementary Figures 2I and3E).

Experiments carried out at the University of California, San Francisco (UCSF) were approved by the institutional animal care and use committee. Mice were bred in specific pathogen-free barrier facilities at UCSF. Pf4-Cre × mTmG mice [START_REF] Lefrançais | The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors[END_REF] were generated from crosses of Pf4-Cre mice (C57BL/6-Tg(Pf4-iCre)Q3Rsko/J, Jackson Laboratories), which express Cre recombinase largely restricted to the megakaryocyte and platelet lineage, [START_REF] Tiedt | Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo[END_REF] with mTmG mice (Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J, Jackson), in which a membrane targeted tandem dimer of tomato fluorescent protein (tdTomato, mT) is constitutively globally expressed, unless Cre recombinase is expressed, where tdTomato gene expression is stopped and expression of membrane-targeted enhanced green fluorescent protein (eGFP, mG) is activated. [START_REF] Muzumdar | A global double-fluorescent Cre reporter mouse[END_REF] Male mice at 8-12 weeks of age were used for intravital microscopy experiments due to their larger size. Terminal anaesthesia was induced with 125 mg/kg ketamine/12.5 mg/kg xylazine (i.p.).

Intranasal challenge with LPS

To induce lung inflammation, mice were briefly anaesthetised with inhaled isoflurane, then challenged with LPS (from E. coli, O55:B5 serotype, Sigma Aldrich 5mg/kg), or PBS vehicle control, by intranasal delivery. Samples (lungs, BAL fluid) were collected 48 hours later as previously described. [START_REF] Pan | P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of in fl ammation[END_REF][START_REF] Ortiz-Muñoz | Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[END_REF] Administration of blocking or depleting antibodies and detection of circulating platelet-neutrophil complexes.

Neutrophil depletion was achieved with three doses of monoclonal anti-Ly6G antibody (clone 1A8, BioXCell, 25 mg/kg i.p.) at -24, -1 and +24 hours relative to intranasal challenge based on previously reported work. [START_REF] Daley | Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice[END_REF] For experimental blockade of adhesion molecule function, either an anti-P-selectin monoclonal antibody (clone RB40.34, BD Biosciences), or an anti-PSGL-1 monoclonal antibody (clone 4RA10 BioXCell), was administered (4 mg/kg i.v. in PBS) at -1 and +24 hours relative to intranasal LPS challenge, with dose and frequency selected based on previously published work investigating the effects of LPS inhalation in mice, [START_REF] Kornerup | Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration[END_REF] and other reports. [START_REF] Sreeramkumar | Neutrophils scan for activated platelets to initiate inflammation[END_REF][START_REF] Phillips | Single injection of P-selectin or P-selectin glycoprotein ligand-1 monoclonal antibody blocks neointima formation after arterial injury in apolipoprotein E-deficient mice[END_REF] Equivalent doses of monoclonal antitrinitrophenol (clone 2A3, BioXCell, trinitrophenol is not present in mammals) were administered to control mice. At 4 hours post challenge, some groups of mice were terminally anaesthetised and whole blood collected via cardiac puncture into citrated syringes for flow cytometric analysis of circulating platelet-neutrophil complexes.

Intravital microscopy

At 48 hours following PBS or LPS inhalation, Pf4-Cre × mTmG mice were anaesthetised. The dorsal left ribcage was shaved, the trachea cannulated and the mouse attached to a Mini Vent ventilator with isoflurane and oxygen supplied at 130× 250 µl breaths per minute with 2 cmH 2 O of positive end expiratory pressure. Skin over the shaved region was removed, and an approximately 1 cm long intercostal incision was made to expose the ventilated left lung and allow the surface of the visceral pleura to fall away from the parietal pleura. Body temperature was maintained using a heated stage (37 °C) and mice administered PBS (0.5ml i.p.) as fluid support.

As previously reported, [START_REF] Lefrançais | The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors[END_REF][START_REF] Looney | Stabilized imaging of immune surveillance in the mouse lung[END_REF] a custom-made thoracic window was then inserted to sit between two ribs, secured onto a 90° angle post clamp attached to the stage and 3-3.5 kPa negative pressure was applied between the visceral pleura of the lung and the inner surface of the window using an Amvex vacuum regulator in order to immobilise the lung, and to remove air from the pleural cavity.

After around 10 minutes of stabilisation, the lung was brought into focus on a custom Nikon A1R multiphoton microscope coupled with a MaiTai DeepSee infrared excitation laser tuned to 920 nm for simultaneous excitation of eGFP and tdTomato, using a x25 objective Nikon water immersion lens, and emission filters set for tdTomato at 570-620 nm and for eGFP at 500-550 nm. Image capture was controlled using NIS Element AR software with high resolution bi-directional Galvano scanning over 512×512 pixels.

The internal surface of the final generation of alveoli, a densely networked capillary bed, was brought into focus, and viability of the mouse confirmed by observation of blood flow in the pulmonary vessels.

Then using a Z-drive the imaging plane was moved 10 µm deeper into the lung to establish an imaging plane at a consistent depth into the lung. Video recordings at the maximum speed possible using Galvano scanning (0.91 frames per second) were then made in order to produce datasets where platelets adhesion in the lung microvasculature could be quantified. For each mouse, 8× videos of 230×230 µm fields with a duration of 5 minutes were sampled at different sites across the window, and videos were screened in a blinded fashion and excluded from analyses if stable imaging was not maintained, or if large blood vessels or megakaryocytes were in the sample region.

Analysis of platelet adhesion using intravital image sequences

Intravital image sequences were analysed using NIS Elements 4.5 (Nikon) tracking. Platelets were identified by size and eGFP fluorescence intensity, and then platelet adhesion events were quantified by tracking platelets that did not move more than 2 µm/s between consecutive frames, whereas freeflowing or transiently adhesive platelets ('non-adhesive') were observed to appear and disappear between frames (Supplementary movie 1).

Experimental design and analysis.

For experiments using blocking or depleting antibody treatment interventions, the experimenter was blinded and randomization within cage blocks was used to assign mice into treatment groups. Other experiments were carried out unblinded, with blinding applied for analysis of samples or images.

Comparisons between two independent groups were made using two-tailed unpaired Student's ttests. Correlations were analysed using Pearson's test to derive the R 2 value describing goodness of fit, with an F-test for the probability that R 2 values from experimental data would be generated from randomly generated datasets with no correlation. Where appropriate, single or repeated measures 1way analysis of variance (ANOVA), 2-way ANOVA, 3-way ANOVA, or three way ANCOVA with baseline values fit as covariates were used. P-values reported are from t-tests, F-tests on Pearson's correlations, or multiplicity-adjusted P-values (Dunnett's or Holm's adjustments where appropriate). The threshold for statistical significance was set at P<0.05.

Data are graphed and reported as means ± standard error. Group sizes (n) are listed in figure legends.

Excel 2016 (Microsoft Office), InVivoStat 3.6 (InVivoStat), and Prism 6.0 (Graphpad).

Results

Platelets and neutrophils are recruited to the lungs after LPS administration in mice.

To record platelet and neutrophil recruitment in the same lung tissue sections of mice administered intranasal LPS, we undertook histological analysis of frozen lung sections stained for CD41 (integrin α-IIb, platelet marker), and neutrophil elastase using immunofluorescence (Figures 1A &1B).

Quantitative analysis of tissue sections revealed a significant increase in pulmonary platelet recruitment in mice administered LPS compared to PBS-administered control mice (Figure 1C), as well as an increase in lung neutrophil elastase staining (Figure 1D). Platelets in lung sections appeared larger with LPS inhalation, although the majority of platelets in sampled fields resembled single platelet events and did not display the morphology of megakaryocytes or thrombi (Supplementary

figure 1A).

LPS inhalation was previously shown to decrease blood platelet counts, [START_REF] Pan | P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of in fl ammation[END_REF] and LPS challenge in the present study also decreased blood platelet counts at 4 hours after challenge, but this thrombocytopaenia was transient, returning to levels observed in control mice at 24 hours after challenge (Figure 1H), providing evidence that changes in platelet numbers in lung sections containing blood at 48 hours post LPS were due to platelet recruitment to lungs rather than changes in numbers of circulating blood platelets.

Immunofluorescence staining allowed for identification of spatial associations of platelets with neutrophil elastase within lung sections (Figure 1E). LPS inhalation increased the quantity of platelets both associated with, and not associated with neutrophil elastase in the lungs (Figure 1F), with an increase in the percentage of platelets that were neutrophil elastase-associated (Figure 1G).

Interventional studies were therefore carried out to further investigate whether interactions of platelets with neutrophils were a causative factor in LPS-induced lung platelet recruitment.

P-selectin and PSGL-1 are dispensable for LPS-induced lung platelet recruitment

Previous observations of the increased presence of neutrophil-associated platelets in the lungs after LPS administration in lung sections (Figures 1F,1G & 1H), and using intravital lung imaging, [START_REF] Pan | P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of in fl ammation[END_REF][START_REF] Ortiz-Muñoz | Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[END_REF] and the requirement of platelets and contact-dependent PSGL-1 interactions for LPS-induced pulmonary neutrophil recruitment, [START_REF] Kornerup | Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration[END_REF] led us to speculate that platelet recruitment might be dependent on the formation of platelet-neutrophil conjugates mediated through PSGL-1. A bright field method was developed for higher-throughput quantification of the platelet marker CD42b (GP1bα, Figure 2A, as well as for neutrophil elastase, Supplementary Figure 1C), and the involvement of P-selectin and PSGL-1 in LPS-induced lung platelet recruitment was tested by administering functional blocking antibodies to mice (Figure 2B).

As reported previously, [START_REF] Kornerup | Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration[END_REF] the administration of an anti-PSGL-1 blocking antibody led to significant and selective reduction in pulmonary neutrophil recruitment to lung airspaces (Figure 2C, Supplementary Figures 2A &2B). Indeed, 4 hours after LPS exposure, the percentage of events that are neutrophils attached to platelets in the circulating leukocyte population was significantly increased in LPS compared to PBS administered mice (P<0.001. Figure 2D), The administration of an anti-PSGL-1 blocking antibody or anti-P-selectin blocking antibody led to a significant reduction in the percentage of events that are neutrophils attached to platelets in the circulating leukocyte population attached to platelets (P<0.05. Figure 2D). In contrast, the presence of platelets in the lungs was not reduced in mice administered either an anti-P-selectin, or anti-PSGL-1 blocking antibody (Figures 2E &2F).

Together, these data suggest that platelet recruitment to lungs following LPS inhalation occurs via a distinct mechanism compared to the mechanisms required for neutrophil recruitment, which show dependence on PSGL-1.

Additionally, neither P-selectin nor PSGL-1 blockade affected the decrease in blood platelet counts at 4 hours following LPS inhalation (Figure 2G), whilst PSGL-1 blockade decreased blood mononuclear cell counts at 4 hours, and blood neutrophil counts at 48 hours; P-selectin blockade increased blood neutrophil counts 48 hours following LPS inhalation (Supplementary Figures 2C-E). The latter effect is a potential mechanism for increases in neutrophil elastase staining in lung sections with anti-P-selectin antibody treatment (Supplementary Figures 2G &2H).

LPS-induced platelet recruitment to lungs is not affected by neutrophil depletion.

To investigate whether pulmonary platelet recruitment was dependent on neutrophils, mice were depleted of circulating neutrophils using an anti-Ly6G antibody (Figure 3A). Anti-Ly6G antibody administration depleted neutrophils from circulating blood in both PBS control mice and in LPSchallenged mice in which inflammation drives greatly enhanced neutrophil production following LPS inhalation over the entire 48 hours (Figure 3B &3E). The anti-Ly6G antibody treatment also reduced neutrophil elastase staining in both control and LPS-challenge lungs (Figures 3C &3D), and reduced neutrophil recruitment to lung airspaces (Figure 3E), with monocyte recruitment to lung airspaces unaffected (Supplementary Figures 3A &3B). Circulating blood mononuclear cells were not affected by the administration of anti-Ly-6G antibody, apart from 4 hours after LPS administration (Supplementary figure 3C &3D), possibly due to the involvement of mononuclear cells in antibodymediated neutrophil clearance. [START_REF] Bruhn | Ly6G-mediated depletion of neutrophils is dependent on macrophages[END_REF] The depletion of neutrophils had no detected effect on lung platelet recruitment (Figures 4A &4B).

Furthermore, LPS administration resulted in an increase in the platelet granule protein PF4 in BAL supernatant, which was not affected by the depletion of neutrophils (Figure 4C). The immunodepletion of neutrophils resulted in a reduction in circulating blood platelet counts, irrespective of, and additional to effects of, LPS administration, at 4 hours, 24 hours and 48 hours (Figure 4D), which could be anticipated to bias towards a finding of decreased lung platelet recruitment in lung sections containing blood. There was a complete absence of CD42b events (platelets) in the lungs of mice depleted of platelets using an anti-platelet depleting antibody, or using the staining protocol in the absence of the primary detection antibody, to signify the robustness of the imaging system and quantification methods, in the absence of a PSGL-1, P-selectin, or neutrophil dependent effect (Supplementary Figure 4).

We also hypothesised that if platelet recruitment was dependent on neutrophil recruitment, mice with high LPS-induced neutrophil recruitment to lungs would have high LPS-induced lung platelet recruitment. Lung platelet (CD42b), and neutrophil elastase staining quantifications from serial sections from all LPS-treated mice were combined (pooled from control mice data in Figures 2E,3D, 4B and 8 additional mice, data shown in Supplementary Figures 5A &5B, shown together in Figures 4F &4G), and tested for correlation. No significant correlation between extent of neutrophil and platelet recruitment was observed (Figure 4H). Additionally, the extent of lung platelet (CD41) and neutrophil elastase staining showed no evidence of any correlation (Supplementary figure 5C, from data in Figures 1C &1D), again providing no evidence supportive of neutrophil dependence of LPSinduced platelet recruitment.

Imaging LPS-induced platelet recruitment in living lungs reveals increased platelet adhesion in the lung microvasculature without platelet aggregation.

In order to further understand the nature of the interactions of platelets with the pulmonary vasculature, and to directly image the effects of LPS inhalation on platelets in the living lung microcirculation, two photon intravital microscopy video recordings were made in lungs of Pf4-Cre × mTmG mice at 48 hours after intranasal exposure to LPS or PBS. The effect of LPS inhalation on the quantity of lung platelet-leukocyte interactions imaged using intravital microscopy has been previously explored using simultaneous genetic labelling of platelets and leukocytes in mice, [START_REF] Ortiz-Muñoz | Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[END_REF] but total platelet recruitment has not previously been measured. Two photon intravital microscopy in Pf4-Cre × mTmG mice, reporter mice with high eGFP fluorophore density on platelet membranes, enabled improved imaging of single platelets passing through the lung microvasculature (Figure 5A), as previously reported. [START_REF] Lefrançais | The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors[END_REF] Image sequences were captured at 0.91 frames per second for 5 minutes from several fields across the visible lung, and tracking software was used to isolate adhesive platelets, which did not move faster that 2 µm/s over 3 or more frames, from platelets which were free-flowing in the bloodstream that appeared and disappeared with each frame (Figure 5B, Supplementary movie 1). LPS inhalation increased the number of adhesive platelets detected in lungs, and no time effect was detected, indicating stable levels of platelet adhesion during acquisition of images (Figures 5C &5D). The mean duration of platelet adhesion events increased, although not significantly after LPS inhalation (Figure 5E). The imaged microvasculature received blood flow during imaging, and nonadhesive platelets were observed in quantities that did not differ between mice challenged with LPS or PBS controls (Figures 5F &5G). It was confirmed that mice used for imaging had raised inflammatory responses to LPS (Figure 5H).

Live imaging revealed that platelet recruitment to the lungs appeared to require vascular adhesion, and platelets did not appear to be attached to neutrophils, although in the microvasculature leukocytes were not easily discriminated from other Pf4-tdTomato+ cells in the lung. Furthermore, platelets appeared as single platelets or small aggregates, and no gross platelet aggregation was evident in the lung microvasculature, and the LPS-induced platelet adhesion events were visually distinct from thromboemboli induced by intravenous infusion of 100 IU/kg human α-thrombin 5I, Supplementary movie 2), or haemostatic thrombosis occurring as a result of incision into the lung surface during intravital window insertion (Figure 5J, Supplementary movie 3). Furthermore the administration of the direct thrombin inhibitor dabigatran etexilate, at a dose sufficient to significantly increase tail bleeding time (Supplementary Figure 6A), and suppress ex vivo platelet aggregation towards thrombin from pooled platelet rich plasma (Supplementary Figure 6B), did not suppress platelet recruitment to the lungs in response to LPS (PBS + vehicle : 320.1±180.0 platelets/mm 2 ; LPS + vehicle: 806.6±314.1 platelets/mm 2 ; LPS + dabigatran: 757.7±490.2 platelets/mm 2 Supplementary Figure 6C. Data not significantly different between groups) or event size (Supplementary Figure 6D. Data not significantly different between groups), suggesting that platelet adhesion was not likely to be dependent on fibrin deposition in the lungs as a result of thrombin activation.

(Enzyme Research Laboratories) (Figure
As previously reported, [START_REF] Lefrançais | The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors[END_REF] occasional megakaryocyte thrombopoeisis events were observed which had an appearance distinct from adhesive platelets, with these large cells captured in small arterioles and moving over minutes to extend proplatelet projections (Figure 5K, Supplementary movie 4), although sample regions and durations required for platelet adhesion tracking were insufficient for quantification of megakaryocyte thrombopoiesis.

Discussion

This is the first report investigating the relationship of neutrophils with lung platelet recruitment in response to bacterial LPS administered via inhalation. We show that despite the close association of recruited platelets with neutrophils in the lungs, ultimately, platelet recruitment showed no dependence on the co-recruitment of neutrophils, nor P-selectin or PSGL-1 mediated interactions classically associated with platelet-dependent neutrophil recruitment. It is possible that platelet interaction with the vascular endothelium after LPS administration is dependent on other adhesive mechanisms that are attributable to platelet adhesion to damaged vessel walls during trauma, for example platelet GPIb interacting with vWF, or platelet GPIIb/IIIa binding to fibrinogen on the vessel wall. [START_REF] Jackson | Platelets and the injured vessel wall-'rolling into action[END_REF][START_REF] Wei | New insights into the haemostatic function of platelets[END_REF][START_REF] Vargo-Szabo | Cell adhesion mechanisms in platelets[END_REF] However, the administration of a direct thrombin inhibitor (dabigatran etexilate) in this LPS model did not affect platelet recruitment, suggestive that fibrin was not an adhesive substrate for platelets under these conditions. Furthermore, the fact that the bleeding time is not different between mice administered LPS compared to PBS suggests that disseminated intravascular coagulation does not occur in this model.

The relationship between platelets and neutrophils during the innate immune response is complex and involves multiple processes. An overriding theme of this relationship is the requirement for platelets to increase the efficiency of neutrophil recruitment to areas of inflammation or infection.

Selectin, and integrin-mediated events provide both an increase in platelet-neutrophil circulating complexes in blood, [START_REF] Kornerup | Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration[END_REF] increased neutrophil rolling and adhesion events on the endothelial surface, [START_REF] Wang | P-selectin primes leukocyte integrin activation during inflammation[END_REF] increased neutrophil intravascular crawling, [START_REF] Sreeramkumar | Neutrophils scan for activated platelets to initiate inflammation[END_REF] and increased neutrophil diapedesis into tissue. [START_REF] Zuchtriegel | Platelets Guide Leukocytes to Their Sites of Extravasation[END_REF] Furthermore, platelets are sometimes found associated with extravascular neutrophils. [START_REF] Ortiz-Muñoz | Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[END_REF][START_REF] Amison | Platelet Depletion Impairs Host Defence to Pulmonary Infection with Pseudomonas aeruginosa in Mice[END_REF] Interestingly, although no evidence was found in the present study for requirement of neutrophils of pulmonary platelet recruitment, we still observed a highly significant increase in the spatial association of platelets with neutrophils in the lungs after LPS administration. Platelets influence neutrophil effector processes in a contact-dependent manner, for example free-radical formation and phagocytosis, [START_REF] Peters | Circulating platelet-neutrophil complexes represent a subpopulation of activated neutrophils primed for adhesion, phagocytosis and intracellular killing[END_REF] transcellular metabolism of pro-inflammatory lipids, [START_REF] Maugeri | Polymorphonuclear leukocyte-platelet interaction: role of P-selectin in thromboxane B2 and leukotriene C4 cooperative synthesis[END_REF] and involvement in NET formation. [START_REF] Clark | Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood[END_REF][START_REF] Caudrillier | Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury[END_REF][START_REF] Kraemer | Novel Anti-bacterial Activities of β-defensin 1 in Human Platelets: Suppression of Pathogen Growth and Signaling of Neutrophil Extracellular Trap Formation[END_REF] It is not known if these neutrophil activation processes occur due to a different population of platelets accessing lung tissue that are not initially associated with intravascular neutrophil recruitment events, but this is something worthy of follow up experiments.

These findings contrast with previous reports studying the effects of anti-Gr1 antibody mediated neutrophil depletion on lung recruitment of radiolabelled platelets following intraperitoneal injections of LPS, [START_REF] Andonegui | Platelets express functional Toll-like receptor-4[END_REF] or following priming with intraperitoneal administration of LPS, followed by intravenous anti-MHCI antibody administration in a model of transfusion-related acute lung injury (TRALI). [START_REF] Looney | Platelet depletion and aspirin treatment protect mice in a two-event model of transfusion-related acute lung injury[END_REF] This may be explained by differences between anti-Ly6G (1A8) and anti-Gr1 (RB6-8C5) antibodies, with the latter depleting Ly6C+ inflammatory monocytes in addition to Ly6G+ neutrophils, [START_REF] Daley | Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice[END_REF] a difference which can have functional consequences bacterial infection models. [START_REF] Shi | Ly6G+ neutrophils are dispensable for defense against systemic Listeria monocytogenes infection[END_REF] In addition, different routes of LPS administration can produce differing inflammatory responses. For example, in 'organ-on-a-chip' models, platelet recruitment to human lung endothelial cells requires epithelial rather than endothelial exposure to LPS, [START_REF] Jain | A primary human lung alveolus-on-a-chip model of intravascular thrombosis for assessment of therapeutics[END_REF] intraperitoneal LPS administration promotes lung neutrophil recruitment as a distant organ affect away from the inflammatory focus in the peritoneum, and the 2-hit model of TRALI where LPS acts as a priming agent involves a rapid intravascular response in the lung in which injury is not dependent on PSGL-1. [START_REF] Looney | Platelet depletion and aspirin treatment protect mice in a two-event model of transfusion-related acute lung injury[END_REF] Furthermore, unlike previous reports, we were unable to detect pulmonary platelet recruitment at early time points (4 hours following administration of LPS, data not shown). [START_REF] Andonegui | Platelets express functional Toll-like receptor-4[END_REF] However, platelet sequestration might be occurring in different tissues at this earlier time point in our model, made evident by thrombocytopaenia at 4 hours post LPS administration.

Intravital imaging experiments in living lungs provide further evidence that platelet activation and recruitment in inflammation contrasts with that in haemostasis and thrombosis, [START_REF] Page | The involvement of platelets in non-thrombotic processes[END_REF][START_REF] Pitchford | Novel uses for anti-platelet agents as anti-inflammatory drugs[END_REF] as platelet adhesion to the pulmonary vascular endothelium occurred as single events in the absence of platelet aggregation. Intravital microscopy experiments have recently demonstrated a novel immune function of platelet adhesion in which platelets migrate on endothelial cells in the infected liver endothelium in order to accumulate fibrinogen-bound bacteria, with this intravascular crawling of lone platelets shown to still occur following neutrophil and monocyte depletion with anti-Gr-1. [START_REF] Gaertner | Migrating Platelets Are Mechano-scavengers that Collect and Bundle Bacteria[END_REF] However, the efficiency of this process in these circumstances is difficult to determine. [START_REF] Gaertner | Migrating Platelets Are Mechano-scavengers that Collect and Bundle Bacteria[END_REF] The convoluted 3dimensional anatomy of the lung capillary network, lung movements during ventilation, and the sterile LPS model prevented measurements of directed platelet migration in the lungs from imaging reported in the present study, but the platelet adhesion observed in lungs following LPS inhalation is indicative that a similar platelet migration response may occur in the inflamed lung. Indeed, inhalation of LPS, [START_REF] Ortiz-Muñoz | Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[END_REF] bacteria, [START_REF] Amison | Platelet Depletion Impairs Host Defence to Pulmonary Infection with Pseudomonas aeruginosa in Mice[END_REF] and other inflammatory insults, [START_REF] Pitchford | Allergen induces the migration of platelets to lung tissue in allergic asthma[END_REF] can promote migration of platelets into airspaces, and platelets are important for limiting lung infection by Klebsiella pneumoniae, [START_REF] De Stoppelaar | Thrombocytopenia impairs host defense in gram-negative pneumonia derived sepsis[END_REF] Streptococcus pneumoniae, [START_REF] Van Den Boogaard | Thrombocytopenia Impairs Host Defense During Murine Streptococcus pneumoniae Pneumonia[END_REF] and Pseudomonas aeruginosa. [START_REF] Amison | Platelet Depletion Impairs Host Defence to Pulmonary Infection with Pseudomonas aeruginosa in Mice[END_REF] The distinct recruitment of platelets from other recruited inflammatory cells might suggest a role for platelets in the inflammatory response in their own right. Whilst it is not yet evident whether direct platelet microbicidal activity is important for host defence, platelets do contain potent microbicidal activity both in vitro and in vivo. [START_REF] Kraemer | Novel Anti-bacterial Activities of β-defensin 1 in Human Platelets: Suppression of Pathogen Growth and Signaling of Neutrophil Extracellular Trap Formation[END_REF][START_REF] Yeaman | Platelets: at the nexus of antimicrobial defence[END_REF] Interventional studies reported here revealed that neutrophil depletion and subsequent LPS inhalation did not cause shock and mortality as reported with intravenous LPS or TNF-α priming followed by intravenous anti-Gr-1 (RB6-8C5) or anti-Ly6G (1A8) antibodies. [START_REF] Abbitt | Antibody ligation of murine Ly-6G induces neutropenia, blood flow cessation, and death via complement-dependent and independent mechanisms[END_REF][START_REF] Tanaka | Stimulation of Ly-6G on neutrophils in LPSprimed mice induces platelet-activating factor (PAF)-mediated anaphylaxis-like shock[END_REF] However, modest decreases in body weight and platelet counts in anti-Ly6G-treated mice in both PBS and LPS-treated mice suggested that some milder adverse effects were present, and were additional to LPS effects. It is possible that these effects in LPS-challenged mice relate to a requirement for neutrophil myeloperoxidase to limit inflammation following exposure to high doses of LPS. [START_REF] Reber | Neutrophil myeloperoxidase diminishes the toxic effects and mortality induced by lipopolysaccharide[END_REF] The findings that blockade of P-selectin increased, whilst PSGL-1 blockade decreased, blood neutrophil counts in the context of lung inflammation are of note for several reasons. First, they are concordant with knockout mouse studies in which genetic deletion of P-selectin increased steady-state blood neutrophil counts, [START_REF] Robinson | Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment[END_REF] but contrast with data from PSGL-1 knockout mice in which baseline neutrophil counts are elevated. [START_REF] Xia | P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow[END_REF] Second, these findings implicate P-selectin and PSGL-1 as potential molecular mediators of blood neutrophil homeostasis under pressure from acute inflammation. Finally, it is possible that anti-PSGL-1 antibodies which have been characterised as having a blocking function, may potentially deplete circulating neutrophils during inflammation. [START_REF] Sreeramkumar | Neutrophils scan for activated platelets to initiate inflammation[END_REF][START_REF] Miner | Separable requirements for cytoplasmic domain of PSGL-1 in leukocyte rolling and signaling under flow[END_REF] To conclude, we report for the first time the lack of neutrophil-dependence of platelet recruitment in lung inflammation following LPS inhalation, a model where lung airspaces are the inflammatory focus and neutrophils are the major cellular infiltrate. The lung platelet recruitment did not depend on Pselectin or PSGL-1-mediated adhesive interactions and involved platelet adhesion in the absence of platelet aggregation. These findings provide further support for a role for platelets in host defence and inflammation in the lungs that is mechanistically separate from neutrophil recruitment, and distinct from platelet adhesion in thrombosis and haemostasis. Fluorescence micrographs were captured using an InCell Analyser 6000 system (GE Healthcare) with a 20× objective, open aperture, excitation/emission wavelength filter sets for AlexaFluor 594: 561/605nm, AlexaFluor 488: 488/525nm and Hoechst 33258: 405/455nm. Image fields were 665.6×665.6µm. Tiled acquisition was used with 15% overlap for stitching and both IR laser autofocus on the slide/section interface, and software autofocus on the Hoechst channel to maintain focus whilst tiling across the entire section.

Immunohistochemistry using bright field DAB reporter.

Immunofluorescence allowed for the simultaneous visualisation of neutrophil elastase, but required the use of an antirat secondary antibody reporter to amplify CD41 immunostaining, which introduced interference in cell depletion and adhesion molecule blocking experiments where mice were dosed in vivo with antibodies raised in rats. A method was developed using CD42b (GP1bα platelet marker) using a new monoclonal antibody raised in rabbits and 3,3'diaminobenzidine (DAB) reporters allowing bright field imaging.

Dried frozen sections of lung tissue were immersed in 4% vol/vol paraformaldehyde (Sigma Aldrich) in PBS and fixed for 15 minutes. Following a tap water wash, endogenous peroxidase activity was blocked and endogenous pigment bleached by immersion in 3% vol/vol hydrogen peroxide in ethanol (Sigma Aldrich). Slides were then blocked as above and anti-CD42b (SP219 clone, Abcam) or anti-neutrophil elastase antibodies were incubated with the sample for 2 hours. After a tap water wash samples were then incubated with biotinylated anti-rabbit IgG antibodies (BA-1000, Vector labs) for 1 hour, washed again and incubated with avidin and biotinylated horseradish peroxidase complex (Vectastain Elite, Vector labs) for 1 hour before a final wash and development with 1.2 mM DAB (Sigma Aldrich) in 0.1 M Tris buffer with 0.03% hydrogen peroxide for 10 minutes, then counterstaining with Gill's no. 2 hematoxylin (Sigma Aldrich) and mounting in DPX.

With 6 transverse sections of middle left lung collected per mouse, using a 20× objective on a DM 2000 LED bright field microscope with DFC295 camera (Leica) and Leica Application Suite acquisition software v4.4, 6× fields of 447×596 μm of the respiratory portion of lung were captured across each section.

Analysis of images of lung sections

Images were analyzed using ImageJ 1.48 (National Institutes of Health). Sample fields consisting of 6× 666×666 µm regions of fluorescent images were chosen in a blinded fashion based on the nuclei channel. The colour deconvolution of leukocytes that are Ly6G hi neutrophil events attached to CD41+ platelet events. The gating strategy is shown in supplementary figure 7.

Administration of dabigatran etexilate for measurement of ex vivo platelet aggregation, in vivo bleeding time, and processing of lungs for histological analysis.

Groups of mice were treated with 75mg/kg Dabigatran Etexilate (Cambridge Bioscience) or vehicle (Saline) control via oral administration at -1 and 24 hours after LPS administration. 48 hours post challenge, mice were anaesthetised with Isoflurane and bleeding times measured by tail tip amputation over 15 minutes. Mice were then culled with whole blood collected via cardiac puncture into citrated syringes, and lungs collected for histology (see below). Platelet rich plasma was isolated from whole blood and pooled according to groups, and stimulated with 1u/ml Thrombin and aggregation measured over 10 minutes via light transmission aggregometry in a shaking plate reader (SpectraMax 340PC, Molecular Devices) at 595nm at 37°C. Lung collection occurred via tracheal cannulation to allow lungs to be inflated in situ with 0.5ml 4% formalin (Sigma, UK), excised and submerged in 4% formalin (Fisher, UK) for 24 hours. Left lobes underwent dehydration and paraffin wax embedding in a tissue processor over a 20 hour automated cycle (Citadel, UK). Lungs were then sectioned to 7 micrometre thickness using a microtome (Microtom, UK) and loaded onto Superfrost Plus slides (Fisher Scientific, UK).

Detecting platelets in lung tissue

For immunohistochemistry (IHC), lung sections were cleared in xylene and rehydrated in decreasing concentrations of ethanol before undergoing blocking in 4% hydrogen peroxide (Honeywell, UK). Sections underwent antigen retrieval in a pressure cooker, using sodium citrate buffer (pH6.0) for 5 minutes at 100 degrees Celsius before using the CD42b staining protocol described above (Immunohistochemistry using bright field DAB reporter) and quantified as described above (Analysis of images of lung sections).

Bronchoalveolar lavage collection and analysis

After terminal anaesthesia, 3 washes of 0.5 ml of PBS were flushed into the lung via a tracheal catheter and retrieved in succession. Samples were diluted 1:2 in Türk's solution for leukocyte counts and 1:2 in PBS for cytospin preparation (Cytospin 2, Thermo Fisher Scientific), subsequently dried and stained with Kwik Diff for leukocyte differential counts.

Figure 1 .

 1 Figure 1. LPS inhalation causes platelet and neutrophil recruitment to the lungs, increased spatial

Figure 2 .

 2 Figure 2. PSGL-1 blockade suppresses LPS-induced lung neutrophil recruitment but does not affect

Figure 3 .

 3 Figure 3. Treatment with anti-Ly6G antibody depletes neutrophils in blood and lungs. (A) Mice were

Figure 4 .

 4 Figure 4. LPS-induced lung platelet recruitment is unaffected by neutrophil depletion. Mice were

Figure 5 .

 5 Figure 5. LPS inhalation increases platelet adhesion in lungs which is distinguishable from lung

Supplementary figure 3 .

 3 Effects of LPS challenge and anti-Ly6G antibody treatment on BAL and blood leukocyte counts, and body weight. Mice were administered either control IgG or anti-Ly6G antibody at -24h, -1h, and +24h in relation to intranasal administration of PBS or LPS (5mg/kg). (A) BAL total leukocyte counts, and (B) BAL mononuclear cell counts. (C) Blood total leukocyte counts and (D) blood mononuclear cell counts. (E) Body weight changes normalised to pre-antibody administration (-24h) baseline values. Means ± standard error, (A & B) n=6 or (C, D & E) n=12. Analysis was (A, B & F) 2-way ANOVA, or (C & D) 3-way ANOVA with repeated measures, or (E) 3-way ANCOVA with repeated measures and baseline values fit as covariates, all followed by Holm's test for LPS and anti-Ly6G treatment effects,*=P<0.05, **=P<0.01, ***=P<0.001.

AJRCMB Articles in Press. Published on 15-February-2019 as 10.1165/rcmb.2018-0182OC Copyright © 2019 by the American Thoracic Society

Acknowledgements. We wish to thank Abhinav Kumar and Val Millar for guidance with imaging at the Stevenage Bioscience Catalyst, and Kyle Marchuk and Adam Fries for support with imaging at the Biological Imaging Development Centre, UCSF.

This research was funded by a Medical Research Council Doctoral Training Grant (MRC-DTP), and associated in vivo strategic skills funds awarded to Dr Simon Pitchford to fund Simon Cleary; Sackler Foundation awards to Professor Clive Page, and travelling fellowships from the British Pharmacological Society and King's College London Physiology Department awarded to Simon Cleary. Work conducted in the laboratory of Professor Mark Looney was supported by NIH grant: NIH R01HL107386. Conflict of interest.

ONLINE SUPPLEMENTARY MATERIAL

LPS-induced lung platelet recruitment occurs independently from neutrophils, PSGL-1, and P-selectin.

Simon J. Cleary, 1 Carl Hobbs, 2 Richard T. Amison, 1 Stephanie Arnold, 1 Blaze G. O'Shaughnessy, 1 Emma Lefrançais, [START_REF] Kornerup | Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration[END_REF] Beñat Mallavia, [START_REF] Kornerup | Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration[END_REF] Mark R. Looney, [START_REF] Kornerup | Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration[END_REF] Clive P. Page, 1 Simon C. Pitchford. 1* 1 Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, King's College London, London, UK. 2 The Wolfson Centre for Age Related Diseases, King's College London, London, UK. [START_REF] Kornerup | Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration[END_REF] University of California San Francisco (UCSF), Department of Medicine, San Francisco, USA

Methods

Collection of mouse lungs for histology

Mice were terminally anaesthetised, the trachea was opened and the diaphragm dissected along the ventral ribcage to collapse the lungs. A tracheal cannula was introduced and the lungs were inflated with 0.5 ml of OCT (optical cutting temperature compound, VWR International). The heart was then excised to stop circulation, the left lung lobe was removed, cross-sectioned in half laterally, placed cut face down in plastic 15×15 mm moulds (VWR International), and immersed with OCT. Samples were then snap frozen in liquid nitrogen and subsequently stored at -80 °C until required for use. Frozen 10 µm cross-sections of tissue (6-9 per mouse) were made using a Model OT cryostat (Bright Instruments) and thaw-mounted onto Superfrost+ slides (VWR International).

Immunohistochemistry using fluorescent reporters.

For immunofluorescence experiments, dried sections were fixed for 15 minutes in 4% paraformaldehyde in PBS, tap water washed, then non-specific binding blocked for 10 minutes using 1% BSA (Fraction V, Sigma Aldrich) in PBS, which was also used as diluent for later reagents. After a tap water wash, rat anti-mouse CD41 primary antibody (clone MWReg30, Abcam) and rabbit anti-mouse/human neutrophil elastase primary antibody (polyclonal ab68672, Abcam) were co-incubated with the sample for 2 hours. Following a PBS wash, anti-mouse IgG conjugated to AlexaFluor 594 (Thermo Fisher Scientific), anti-rabbit IgG conjugated to AlexaFluor 488 (Thermo Fisher Scientific) and Hoechst 33258 DNA stain (Sigma Aldrich) were incubated together on the sample in 1% BSA in PBS for 1 hour before a final PBS wash.

Slides were mounted in Fluoromount G (Southern Biotech) and stored in the dark at 4°C overnight before imaging. http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html) was used to split the DAB stain and haematoxylin stain colour vectors from RGB images into separate greyscale images, as previously described. 1 Positive staining was defined using an intensity threshold based on no primary control and positively stained sections (typically intensities >150 of 255 on an 8 bit depth). In order to reduce single pixel noise, for platelet counts a size filter was used to remove objects smaller than 0.45 µm 2 .

ImageJ particle analysis was then used to count the number of platelets per mm 2 of section imaged, as well as the percentage of sample covered by positive staining. During inflammation, neutrophil elastase is released from neutrophils, and neutrophils form bundles during transendothelial migration, so cell segmentation was not possible, and this antigen was quantified using only the percentage of sample fields stained positive.

Spatial association of platelet CD41 immunostaining with neutrophil elastase was investigated using the relate objects module of CellProfiler to make separate counts of CD41+ platelets with any overlap with neutrophil elastase staining and those that did not (CellProfiler cell image analysis software, Broad Institute, http://cellprofiler.org).

Leukocyte and platelet counts from blood microsamples

Mice were placed in a surgical recovery chamber at 37 °C for 10 minutes before a 1-2 mm cut was made with a scalpel blade across the lateral tail vein. Using a micropipette, 2 µl of blood was rapidly sampled and diluted 1:100 into Stromatol (Mascia Brunelli) solution for platelet counts, a further 2 µl diluted 1:50 in Türk's solution (Merck) for leukocyte counts, and 4 µl was taken for a blood smear. Platelet and leukocyte counts were made using hemocytometers and smears were stained with Kwik-Diff stain (Thermo-Fisher) for leukocyte differential counts (200 cells identified per smear).

Flow cytometric analysis of Platelet-leukocyte complex formation.

4 hours post challenge, mice were terminally anaesthetised and whole blood collected via cardiac puncture into citrated syringes. Whole blood was then incubated with an anti-CD41-fluorescein isothiocyanate (FITC) monoclonal antibody for platelets and anti-Ly6G-phycoerythin (PE) monoclonal antibody for neutrophils in saturating concentrations for 20 minutes, followed by lysis of red blood cells. Samples were then analysed on a flow cytometer (FC500; Beckman Coulter) as previously described. 2 CD41+ events were gated on Ly-6G hi neutrophils. The percentage