Ronan Kapetanovic
email: r.kapetanovic@imb.uq.edu.au

Syeda Farhana Afroz

Divya Ramnath

Mep Grace

Takashi Lawrence

James Eb Okada

Jost Curson

David P De Bruin

Kate Fairlie

Justin C Schroder

John St

Grace Mep Lawrence

Takashi Okada

James Eb Curson

Jost De Bruin

David P Fairlie

Kate Schroder

Justin C St

Antje Blumenthal

Matthew J Sweet
email: m.sweet@imb.uq.edu.au

Lipopolysaccharide promotes Drp1-dependent mitochondrial fission and associated inflammatory responses in macrophages

Keywords: Dynamin-related protein 1, lipopolysaccharide, macrophage, mitochondrial dynamics, mitochondrial fission, Toll-like receptor

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INTRODUCTION

Innate immune cells such as macrophages act as dangersensing sentinels, responding to environmental perturbations to ensure maintenance of homeostasis. These cells use pattern recognition receptors such as Tolllike receptors (TLRs) to recognize and respond to an array of endogenous molecules released during cellular stress, as well as microbial products such as lipopolysaccharide (LPS). [START_REF] Kawasaki | Toll-Like Receptor Signaling Pathways[END_REF] Functional consequences of TLR activation encompass production of mediators that orchestrate inflammation (e.g. cytokines and chemokines) and expression of molecules that sculpt adaptive immunity (e.g. costimulatory molecules). TLRs signal through the activation of canonical Toll/interleukin-1 (IL-1) receptor-containing adaptor proteins [e.g. myeloid differentiation primary response 88 (MyD88), MyD88 adaptor-like (MAL), Toll/IL-1R domain-containing adaptor-inducing interferon-b (TRIF) and TRIF-related adaptor molecule (TRAM)] [START_REF] Kawasaki | Toll-Like Receptor Signaling Pathways[END_REF] as well as noncanonical non-Toll/IL-1 receptor adaptors (e.g. SCIMP). [START_REF] Luo | SCIMP is a transmembrane non-TIR TLR adaptor that promotes proinflammatory cytokine production from macrophages[END_REF] With respect to Toll/IL-1 receptor-containing adaptors, LPS signaling via TLR4 engages both the MAL/MyD88 and the TRAM/TRIF signaling arms. In the MyD88dependent pathway, MyD88 interacts with other death domain-containing proteins, such as IL-1 receptorassociated kinase 4 and 1, forming an oligomeric complex referred to as the Myddosome. [START_REF] Balka | Understanding early TLR signaling through the Myddosome[END_REF] In addition to triggering transcription factor activation for inducible gene expression, downstream TLR signaling components can also translocate to mitochondria to reprogram their functions. For example, TLR activation results in increased mitochondrial reactive oxygen species generation in macrophages. [START_REF] West | TLR signalling augments macrophage bactericidal activity through mitochondrial ROS[END_REF] Further links between TLRs and mitochondria have emerged through discoveries showing that TLR signaling triggers major changes in metabolic pathways in murine macrophages. [START_REF] Ryan | Krebs cycle rewired for macrophage and dendritic cell effector functions[END_REF] Hallmarks of this remodeling are increased glycolysis and flux through the tricarboxylic acid cycle to generate acetyl coenzyme A for inducible gene expression. [START_REF] Lauterbach | Toll-like receptor signaling rewires macrophage metabolism and promotes histone acetylation via ATP-citrate lyase[END_REF] At later time points, the tricarboxylic acid cycle is disrupted, leading to the accumulation of tricarboxylic acid cycle intermediates such as succinate that have been implicated in specific inflammatory responses. [START_REF] Tannahill | Succinate is an inflammatory signal that induces IL-1b through HIF-1a[END_REF] Mitochondria are incredibly dynamic organelles; under different conditions, they can exist as a complex network (driven by fusion) or as fragmented organelles (driven by fragmentation/fission). [START_REF] Giacomello | The cell biology of mitochondrial membrane dynamics[END_REF] For example, severe cellular stress and impaired oxidative phosphorylation are associated with mitochondrial fission, whereas nutrient starvation and increased oxidative phosphorylation are linked to mitochondrial fusion. [START_REF] Wai | Mitochondrial dynamics and metabolic regulation[END_REF] Mitochondrial dynamics are controlled by several dynamin-related GTPases, including dynamin-related protein 1 (DRP1), mitofusins 1 and 2, and optic atrophy 1. [START_REF] Giacomello | The cell biology of mitochondrial membrane dynamics[END_REF] While the mitofusins and specific optic atrophy 1 isoforms promote mitochondrial tubulation, [START_REF] Song | OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L[END_REF] DRP1 is essential for the fission response. [START_REF] Smirnova | Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells[END_REF] Cytoplasmic DRP1 is recruited by mitochondrial fission factor [START_REF] Otera | Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells[END_REF] and other mitochondrial proteins [START_REF] Loson | Fis1, Mff, MiD49, and MiD51 mediate Drp1 recruitment in mitochondrial fission[END_REF] to the mitochondrial outer membrane in combination with the endoplasmic reticulum, where it promotes fission by constricting the mitochondria. [START_REF] Rowland | Endoplasmic reticulummitochondria contacts: function of the junction[END_REF] DRP1 activity is regulated by several post-translational modifications, with phosphorylation at S616 (mouse S635) [START_REF] Taguchi | Mitotic phosphorylation of dynamin-related GTPase Drp1 participates in mitochondrial fission[END_REF] and dephosphorylation at S637 (mouse S656) [START_REF] Chang | Cyclic AMP-dependent protein kinase phosphorylation of Drp1 regulates its GTPase activity and mitochondrial morphology[END_REF][START_REF] Cereghetti | Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria[END_REF][START_REF] Yu | Dephosphorylation by calcineurin regulates translocation of dynamin-related protein 1 to mitochondria in hepatic ischemia reperfusion induced hippocampus injury in young mice[END_REF] being particularly implicated in its regulation. The exact roles of these and other DRP1 modifications in its activation or inhibition are likely to be cell type and signal dependent.

Previous studies have provided some biochemical [START_REF] Katoh | Polymorphic regulation of mitochondrial fission and fusion modifies phenotypes of microglia in neuroinflammation[END_REF][START_REF] Nair | Lipopolysaccharideinduced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo[END_REF] and genetic [START_REF] Park | Defective mitochondrial fission augments NLRP3 inflammasome activation[END_REF][START_REF] Park | Mitochondrial dynamics modulate the expression of pro-inflammatory mediators in microglial cells[END_REF] evidence to support a role for LPS in regulating mitochondrial dynamics. However, another study reported that LPS stimulates mitochondrial biogenesis in macrophages, [START_REF] Zhong | New mitochondrial DNA synthesis enables NLRP3 inflammasome activation[END_REF] and the relationship between the effects of LPS on mitochondrial numbers, mitochondrial fission and mitochondrial biogenesis remains unclear. Moreover, little is known about the specific receptors and downstream signaling pathways associated with LPS-inducible fission. Here, we address these questions, providing compelling evidence for a Tlr4-MyD88-Drp1 axis as a mediator of LPS-inducible fission in macrophages and of sequential Drp1 activation during this process. Our study also adds further weight to the emerging concept that mitochondrial fission is linked to specific inflammatory responses. [START_REF] Joshi | Fragmented mitochondria released from microglia trigger A1 astrocytic response and propagate inflammatory neurodegeneration[END_REF][START_REF] Wang | Inhibition of DNM1L and mitochondrial fission attenuates inflammatory response in fibroblast-like synoviocytes of rheumatoid arthritis[END_REF]

RESULTS

LPS triggers Tlr4-and MyD88-dependent mitochondrial fission in macrophages

We began by examining mitochondrial morphology in bone marrow-derived macrophages (BMMs) following LPS stimulation. LPS appeared to promote mitochondrial fragmentation (Figure 1a, b), which was quantified by assessing mitochondrial numbers with two independent methods in an unbiased fashion, the maxima method (assessing the maximum intensity point; Figure 1c, Supplementary figure 1a) and the skeletal method (assessing the connectivity of the mitochondrial network; Figure 1d, Supplementary figure 1b). Because of the consistency in the maxima method across different experiments, this method of quantification was used for all subsequent analyses. The specificity of MitoTracker staining was demonstrated by costaining for the mitochondrial proteins heat-shock protein 60 and Tom20 (Supplementary figure 1c,d). Some LPS responses, including with respect to signaling, [START_REF] Sun | Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use[END_REF] gene expression [START_REF] Schroder | Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages[END_REF] and cell metabolism, [START_REF] Vijayan | Human and murine macrophages exhibit differential metabolic responses to lipopolysaccharide-a divergent role for glycolysis[END_REF] are divergent between primary mouse and human macrophages. Thus, we next assessed LPSinducible mitochondrial fragmentation in human monocyte-derived macrophages. Here we found that, as observed in BMMs, LPS increased mitochondrial numbers in these cells (Figure 1e, Supplementary figure 1e,f). Given this conservation in response between human and mouse macrophages, we next used BMMs to explore molecular mechanisms involved. We first investigated whether the effect of LPS is mediated by its transmembranal receptor Tlr4 [START_REF] Poltorak | Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene[END_REF] or the cytosolic LPS receptor caspase-11. [START_REF] Hagar | Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock[END_REF][START_REF] Kayagaki | Noncanonical inflammasome activation by intracellular LPS independent of TLR4[END_REF] Here we found that the LPS-mediated increase in mitochondrial numbers was defective in Tlr4 À/À BMMs, but not in Casp1/ 11 À/À BMMs (Figure 1f). Moreover, the response was absolutely dependent on the TLR adaptor protein MyD88 (Figure 1g).

(i) (j) (k) (l) (f) (g) (h) MitoTracker MyD88 -/- Casp1/11 -/- Tlr4 -/- M1 + mdivi1:

MitoTracker

Figure 1. LPS triggers Tlr4-and MyD88-dependent mitochondrial fission in macrophages. (a-l) Unless otherwise indicated, BMMs were stimulated with LPS (100 ng mL À1 ) or left untreated for 6 h, prior to performing indicated analyses. Mitochondrial numbers were determined relative to the unstimulated control in each experiment. (a) BMMs were stained with MitoTracker (mitochondria, red) and DAPI (nuclear DNA, blue). (b) The mitochondrial network was reconstituted in 3D using Imaris software. (c, d) Relative mitochondrial number observed in BMMs using (c) a maxima analysis method and (d) a skeletal analysis method (same data sets used for each analysis). Data are combined from four independent experiments (mean AE s.e.m., one sample t-test). (e) Relative mitochondrial numbers in human monocyte-derived macrophages (mean AE s.e.m., three independent experiments, one sample t-test). (f) Relative mitochondrial numbers in WT, Tlr4 À/À and Caspase 1/11 À/À BMMs [mean AE s.e.m., four independent experiments, two-way ANOVA with Sid ak's multiple comparisons test]. (g) Relative mitochondrial numbers in WT and MyD88 À/À BMMs (mean AE s.e.m., three independent experiments, two-way ANOVA with Sid ak's multiple comparisons test). (h) Median fluorescence intensity (MitoTracker) per cell (mean AE s.e.m., six independent experiments, t-test). (i) Mitochondrial protein Tom20 levels (relative to tubulin) in BMMs stimulated with LPS for the indicated times. Cells were lysed, after which Tom20 and tubulin levels were assessed by western blot (mean AE s.e.m., four independent experiments, one-way ANOVA with Dunnett's multiple comparisons test). (j, k) BMMs from male (gray circles) and female (red circles) mice were treated with LPS for 3, 6 or 24 h. (j) Tnf production was assessed by ELISA and (k) mitochondrial DNA copy number relative to nuclear DNA was determined (mean AE s.e.m., BMMs harvested from eight different mice, oneway ANOVA with Dunnett's multiple comparisons test). (l) BMMs were pretreated for 1 h with DMSO or M1 + mdivi1, after which cells were stimulated with LPS and relative mitochondrial numbers were calculated (mean AE s.e.m., five independent experiments, two-way ANOVA with Sid ak's multiple comparisons test). In all panels, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Scale bars, 5 µm. BMMs, bone marrowderived macrophages; Con, control; DAPI, 4 0 ,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; LPS, lipopolysaccharide; MFI, mean fluorescent intensity; ns, not significant; Tnf, tumor necrosis factor; WT, wild type.

We next considered if the increase in mitochondrial numbers was linked to increased mitochondrial biogenesis and/or reduced mitophagy. The median fluorescence intensity of MitoTracker-stained cells was similar in control and LPS-treated cells (Figure 1h) and LPS treatment did not alter Tom20 protein levels (Figure 1i, see also Figure 3). This suggests that the mitochondrial mass is comparable in control and LPStreated BMMs. Next, we assessed the effect of LPS on mitochondrial DNA copy number in BMMs. Whereas LPS induced tumor necrosis factor (TNF) production in BMMs (Figure 1j), the ratio of mitochondrial genes (non-NUMT, a specific region of mitochondrial DNA) [START_REF] Zhong | New mitochondrial DNA synthesis enables NLRP3 inflammasome activation[END_REF] to nuclear genes (nuclear DNA B2m) remained unchanged over an LPS time course (Figure 1k). Collectively, these data (Figure 1h-k) suggest that, although LPS induces fragmentation of the mitochondrial network, it does not increase mitochondrial mass and/or mitochondrial DNA replication. Finally, we further validated our methodology by confirming that the mitochondrial fusion-promoting compound M1 [START_REF] Wang | A small molecule promotes mitochondrial fusion in mammalian cells[END_REF] in combination with the Drp1 inhibitor mdivi1 [START_REF] Cassidy-Stone | Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization[END_REF] blocked the LPS-inducible increase in mitochondrial numbers in BMMs (Figure 1l). We conclude that LPS acts via Tlr4 and MyD88 to skew toward mitochondrial fission in macrophages.

LPS-inducible mitochondrial fragmentation is dependent on Drp1

To further confirm that LPS induces mitochondrial fission, we next assessed the involvement of the fissionpromoting GTPase Drp1 using genetic approaches. Both small interfering RNA-mediated Drp1 silencing in BMMs (Supplementary figure 2a,b) and Drp1 deletion in mouse embryonic fibroblast (MEF) cells [START_REF] Wakabayashi | The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice[END_REF] (Supplementary figure 2c) revealed that LPS-induced mitochondrial fragmentation required Drp1 (Figure 2a,b). This was further confirmed by quantifying LPS-inducible mitochondrial numbers in these cells (Figure 2c,d). Drp1 is recruited from the cytoplasm to mitochondria to initiate mitochondrial fission, [START_REF] Osellame | Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission[END_REF] and we also observed that LPS increased the association of Drp1 with mitochondria (Figure 2e, f, Supplementary figure 2d). Thus, LPS promotes Drp1 recruitment to mitochondria to initiate fission.

LPS regulates Drp1 phosphorylation in BMMs

We next examined LPS-regulated post-translational modifications on Drp1. Specific post-translational modifications on Drp1, particularly phosphorylation of S616 [START_REF] Taguchi | Mitotic phosphorylation of dynamin-related GTPase Drp1 participates in mitochondrial fission[END_REF] (S585 in rat, S635 in mouse) and dephosphoryla-tion of S637 (S656 in mouse) [START_REF] Chang | Cyclic AMP-dependent protein kinase phosphorylation of Drp1 regulates its GTPase activity and mitochondrial morphology[END_REF][START_REF] Cereghetti | Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria[END_REF][START_REF] Yu | Dephosphorylation by calcineurin regulates translocation of dynamin-related protein 1 to mitochondria in hepatic ischemia reperfusion induced hippocampus injury in young mice[END_REF] (Figure 3a), have been associated with its activation as well as its translocation to the mitochondria in different cell types. In BMMs, LPS promoted acute phosphorylation (approximately 15-60 min after stimulation) of Drp1 at S635, whereas the levels of total Drp1 remained unchanged (Figure 3b,c). LPS also triggered dephosphorylation of Drp1 at S656, with this effect being apparent at approximately 6 h after stimulation and being sustained for 24 h (Figure 3d,e). Because Drp1 phosphorylation at S635 was a rapid response, we next examined mitochondrial fission across an LPS time course. Indeed, a trend for increased mitochondrial numbers was apparent as early as 1 h after LPS stimulation, with the response plateauing at approximately 6-9 h after LPS treatment (Figure 3f). To gain further insight into post-translational modifications on Drp1 that are likely important for LPS-inducible fission, we monitored the Drp1 phosphorylation status in MyD88 À/À BMMs, because the LPS-inducible mitochondrial fission response was defective in these cells (Figure 1g). Whereas the transient phosphorylation of Drp1 on S635 still occurred in MyD88 À/À BMMs, S656 dephosphorylation was abrogated (Figure 3g, Supplementary figure 2e,f). Thus, LPS triggers rapid Drp1 phosphorylation at S635 independently of MyD88, whereas dephosphorylation of Drp1 at S656 requires this TLR adaptor.

Mitochondrial fission regulates a subset of Tlr4inducible inflammatory responses

Some studies have linked mitochondrial dynamics to inflammatory responses, particularly in microglial cells. [START_REF] Nair | Lipopolysaccharideinduced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo[END_REF][START_REF] Park | Mitochondrial dynamics modulate the expression of pro-inflammatory mediators in microglial cells[END_REF] Thus, we next investigated whether LPSinducible mitochondrial fission regulates inflammatory responses in BMMs. Pretreating BMMs with M1 and mdivi1 that skew mitochondrial dynamics toward fusion [START_REF] Wang | A small molecule promotes mitochondrial fusion in mammalian cells[END_REF][START_REF] Cassidy-Stone | Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization[END_REF] resulted in reduced LPS-inducible production of the proinflammatory cytokines Il-12p40 and Il-6 (Figure 4a,b). These effects were selective, as the combination of M1 and mdivi1 did not affect inducible production of nitric oxide in BMMs (Figure 4c). The inhibitory effect on Il-12p40 and Il-6 in BMMs was also apparent at the messenger RNA (mRNA) level (Figure 4d, e), whereas the marginal reduction of LPSinducible Nos2 mRNA (required for nitric oxide production) was not statistically significant (Figure 4f). LPS-inducible Ccl2 was also unaffected (Supplementary figure 3a), confirming that M1/mdivi1 did not globally affect LPS responses in BMMs. Given that mdivi1 can have off-target effects, [START_REF] Bordt | The Putative Drp1 Inhibitor mdivi-1 Is a Reversible Mitochondrial Complex I Inhibitor that Modulates Reactive Oxygen Species[END_REF] we next examined Drp1 dependence for LPS responses using Drp1-deficient MEF cells. In this system, we examined LPS-regulated gene expression, because levels of secreted Il-12p40 and Il-6 were too low for accurate quantification. Here we found that Drp1 deletion abrogated LPS-inducible Il12b mRNA expression (Figure 4g), with a trend toward reduced Il6 (Figure 4h). By contrast, Nos2 and Ccl2 were not significantly affected (Figure 4i, Supplementary figure 3b). The proinflammatory mediators Il-1b and Tnf are regulated not only by transcriptional and translational mechanisms, but also by post-translational cleavage events. Tnf is produced as a transmembranal precursor that is cleaved by the metalloproteinase TNF-aconverting enzyme, [START_REF] Black | A metalloproteinase disintegrin that releases tumour-necrosis factor-a from cells[END_REF] whereas release of Il-1b from cells requires inflammasome-dependent cleavage by proinflammatory caspases. [START_REF] Chan | Inflammasome signaling and regulation of interleukin-1 family cytokines[END_REF] Experimentally, the microbial product nigericin is widely used to initiate inflammasome activation and processing of pro-Il-1b for secretion of bioactive Il-1b. Here we found that antagonizing fission significantly reduced nigericin-triggered Il-1b release from LPS-primed BMMs (Figure 4j), whereas LPS-inducible Il1b mRNA expression was only modestly affected in the same cells (Supplementary figure 3c). Similarly, there was a clear trend for M1/mdivi1 to reduce LPS-induced Tnf secretion in BMMs (Figure 4k), with only a very minor effect on Tnf mRNA levels being apparent (Supplementary figure 3d). We conclude that LPS triggers Drp1 activation for inducible fission and inflammatory responses in macrophages, possibly through multiple mechanisms.

DISCUSSION

LPS is a potent regulator of metabolism and mitochondrial functions in macrophages, [START_REF] Tannahill | Succinate is an inflammatory signal that induces IL-1b through HIF-1a[END_REF] but only a limited number of studies have explored LPS-regulated mitochondrial fission in this cell type. [START_REF] Katoh | Polymorphic regulation of mitochondrial fission and fusion modifies phenotypes of microglia in neuroinflammation[END_REF][START_REF] Nair | Lipopolysaccharideinduced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo[END_REF][START_REF] Park | Mitochondrial dynamics modulate the expression of pro-inflammatory mediators in microglial cells[END_REF][START_REF] Baker | Molecular and cellular mechanisms responsible for cellular stress and low-grade inflammation induced by a super-low dose of endotoxin[END_REF] Here we

532

build on these studies, profiling LPS-inducible fission in both primary human and mouse macrophages, examining other important parameters such as mitochondrial DNA synthesis in parallel, defining signaling components that are required for the response and using complementary genetic and pharmacological approaches to validate key findings. In so doing, we provide compelling evidence of LPS-inducible mitochondrial fission in macrophages, delivering new insights into molecular mechanisms involved and the downstream consequences. We note, however, that although we have demonstrated that LPS increases mitochondrial fragmentation and numbers in human monocyte-derived macrophages, we did not examine molecular mechanisms of inducible fission in these cells. Given known differences between human and murine macrophages responding to LPS, [START_REF] Sun | Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use[END_REF][START_REF] Schroder | Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages[END_REF] such studies are clearly warranted. Furthermore, LPS-inducible glycolysis has been reported to be dependent on Drp1 and/or fission (discussed later), yet LPS did not increase glycolysis in human macrophages. [START_REF] Vijayan | Human and murine macrophages exhibit differential metabolic responses to lipopolysaccharide-a divergent role for glycolysis[END_REF] Thus, the functional consequences of LPS-inducible fission in human macrophages also require further investigation.

In investigating underlying molecular mechanisms in mouse BMMs, we found that both Tlr4 and MyD88 are required for LPS-inducible mitochondrial fission. A previous study reported that a low dose of LPS induced mitochondrial fragmentation in BMMs via an IL-1 receptor-associated kinase 1-dependent mechanism. [START_REF] Baker | Molecular and cellular mechanisms responsible for cellular stress and low-grade inflammation induced by a super-low dose of endotoxin[END_REF] Our findings are consistent with this study, because IL-1 receptor-associated kinase 1 lies downstream of MyD88 in the Tlr4 pathway. [START_REF] Wesche | MyD88: an adapter that recruits IRAK to the IL-1 receptor complex[END_REF] We also show that LPS triggers sequential post-translational modifications on Drp1, namely transient phosphorylation on S635 and sustained dephosphorylation on S656. Unlike S635 phosphorylation, S656 dephosphorylation was dependent on MyD88, implying that this modification is likely to be particularly important for initiating LPS-inducible mitochondrial fission. Phosphorylation of S616 on human DRP1 (corresponding to mouse S635) by either cyclin B/Cdk1 or extracellular signal-regulated kinase 2 was linked to the recruitment of DRP1 to the mitochondria and the initiation of fission. [START_REF] Kashatus | RALA and RALBP1 regulate mitochondrial fission at mitosis[END_REF][START_REF] Kashatus | Erk2 phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth[END_REF] The acute LPS-inducible phosphorylation of S635 observed here did not require MyD88, so the role of this modification in macrophage responses to LPS remains to be determined. It may be involved in the early increase in mitochondrial numbers that we observed here (1-2 h after LPS stimulation). Another possibility is that it is involved in LPS-inducible inflammatory responses independently of fission. This may be important, given that some studies on LPS-inducible fission have focused on regulation of S616/S635. [START_REF] Katoh | Polymorphic regulation of mitochondrial fission and fusion modifies phenotypes of microglia in neuroinflammation[END_REF][START_REF] Zhang | Lipopolysaccharideinduced proliferation and glycolysis in airway smooth muscle cells via activation of Drp1[END_REF] In contrast to inducible S635 phosphorylation, dephosphorylation of S656 did require MyD88, implicating this post-translational modification in the inducible fission response. Because S656 dephosphorylation only occurred several hours after LPS stimulation, it is likely that autocrine/paracrine factors and/or regulated expression of phosphatases and/or kinases that modify this residue may be important. Calcineurin and cyclic adenosine monophosphatedependent protein kinase, which both modulate the phosphorylation status of S656 in other cell types, [START_REF] Chang | Dynamic regulation of mitochondrial fission through modification of the dynaminrelated protein Drp1[END_REF] are also implicated in LPS responses. [START_REF] Suzuki | Lipopolysaccharide activates calcineurin in ventricular myocytes[END_REF][START_REF] Wall | Suppression of LPS-induced TNF-a production in macrophages by cAMP is mediated by PKA-AKAP95-p105[END_REF] Furthermore, a role for calcineurin in LPS-mediated Drp1 dephosphorylation in microglia has previously been demonstrated. [START_REF] Park | Peroxiredoxin 5 (Prx5) decreases LPS-induced microglial activation through regulation of Ca 2+ /calcineurin-Drp1-dependent mitochondrial fission[END_REF] These therefore represent potential candidates for regulating

TLR-driven Drp1 dephosphorylation and fission in

BMMs and other macrophage populations. In addition, several other unknowns around the signaling framework for LPS-inducible fission in macrophages remain. Specifically, whether S635 phosphorylation and/or S656 dephosphorylation have causal roles in LPS-inducible fission in macrophages is yet to be determined. This could be interrogated in the future through reconstitution of Drp1-deficient cells with S635A and/or S656E Drp1 mutants. Roles for other TLR adaptor molecules such as TRIF and TRAM, particularly in acute S635 phosphorylation and the early fission response, also need to be addressed. Whereas our data clearly show that LPS induces mitochondrial fission in BMMs, we found no evidence of increased mitochondrial DNA replication or biogenesis in these cells. We note that we examined the effect of LPS on mitochondrial DNA copy number over a time course in BMMs from both male and female mice. Our findings contrast with those of Zhong et al. who also studied BMMs, finding that LPS increased mitochondrial DNA content and that this primed inflammasome responses. [START_REF] Zhong | New mitochondrial DNA synthesis enables NLRP3 inflammasome activation[END_REF] The reasons for the different observations in this study versus our own are unclear, but might be related to variation in cell culture conditions. Although both studies used similar concentrations of LPS and glucose in culture media, there were differences in other variables such as the macrophage differentiation methods. Whatever the explanation, our study clearly shows that LPS-inducible fission can occur in the absence of inducible mitochondrial DNA synthesis in macrophages. This might also be true in human disease, as it was shown that DRP1 protein expression was elevated in tissues of critically ill patients versus those of controls, whereas there was no difference in mitochondrial DNA copy number between these groups. [START_REF] Vanhorebeek | Mitochondrial fusion, fission, and biogenesis in prolonged critically ill patients[END_REF] Our findings are also somewhat consistent with a study in PC-12 cells where rotenone promoted mitochondrial fission but actually impaired mitochondrial biogenesis via the transcription factor peroxisome proliferator-activated receptor gamma coactivator 1-alpha. [START_REF] Peng | The Interaction of Mitochondrial Biogenesis and Fission/Fusion Mediated by PGC-1a Regulates Rotenone-Induced Dopaminergic Neurotoxicity[END_REF] We suggest that the LPS-induced increase in mitochondrial numbers without a corresponding increase in mitochondrial DNA copy number might be analogous to a cell that has a greater abundance of na€ ıve mitochondria. This occurs in oocytes, for example, where there is less dependence on oxidative phosphorylation, matched by fewer copies of the mitochondrial genome per mitochondrion. [START_REF] Piko | Number of mitochondria and some properties of mitochondrial DNA in the mouse egg[END_REF][START_REF] Piko | Amounts of mitochondrial DNA and abundance of some mitochondrial gene transcripts in early mouse embryos[END_REF] In this scenario, LPS-induced fission in macrophages might be predicted to result in a decrease in the average number of mitochondrial genomes per mitochondrion. Future studies should examine the relationship between inducible fission and mitochondrial DNA synthesis in activated macrophages in vivo in different inflammatory contexts.

In both smooth muscle cells [START_REF] Zhang | Lipopolysaccharideinduced proliferation and glycolysis in airway smooth muscle cells via activation of Drp1[END_REF] and microglia, 20 mitochondrial fission has been implicated in LPSinducible glycolysis. This metabolic response is known to be required for the production of a subset of inflammatory mediators, [START_REF] Ryan | Krebs cycle rewired for macrophage and dendritic cell effector functions[END_REF] thus implying that fission may promote inflammatory responses. Consistent with this, we found that genetic as well as pharmacological targeting of fission reduced the production of several proinflammatory cytokines, including Il-12p40, Il-6, Tnf and Il-1b. These findings are broadly consistent with other studies undertaken in microglia. [START_REF] Nair | Lipopolysaccharideinduced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo[END_REF][START_REF] Park | Mitochondrial dynamics modulate the expression of pro-inflammatory mediators in microglial cells[END_REF] Interestingly, the glycolytic enzyme pyruvate kinase 2 isoform M2 was shown to modulate mitochondrial dynamics via an interaction with mitofusin 2. [START_REF] Li | PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation[END_REF] This interaction promoted mitochondrial fusion and controlled the metabolic switch between glycolysis and oxidative phosphorylation in cancer. [START_REF] Li | PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation[END_REF] Pyruvate kinase 2 isoform M2 also drives LPSinducible Il-1b production in murine macrophages, [START_REF] Palsson-Mcdermott | Pyruvate kinase M2 regulates Hif-1a activity and IL-1b induction and is a critical determinant of the warburg effect in LPS-activated macrophages[END_REF][START_REF] Gupta | Class IIa histone deacetylases drive toll-like receptor-inducible glycolysis and macrophage inflammatory responses via pyruvate kinase M2[END_REF] a cytokine we found to be downstream of LPS-induced fission. One possibility is that LPS-induced fission inhibits the fusion-promoting activity of pyruvate kinase 2 isoform M2, enabling it to perform its inflammatory functions. Thus, it is plausible that glycolysis drives LPSinducible Il-1b production as a consequence of increased mitochondrial fission. We also observed that Il-12p40 production was particularly dependent on the mitochondrial fission response, with LPS-inducible Il12b mRNA expression being abrogated by Drp1 deletion in MEF cells. Consistent with our data, Gao et al. found that promoting mitochondrial fission by silencing Fam73b increased IL-12 production in macrophages. [START_REF] Gao | Mitochondrial dynamics controls anti-tumour innate immunity by regulating CHIP-IRF1 axis stability[END_REF] Thus, future studies on Il12b regulation are likely to provide insights into the molecular mechanisms by which Drp1 and/or mitochondrial fission promote macrophage inflammatory responses. Mitochondrial fission was recently linked to sepsis-induced cardiomyopathy. [START_REF] Haileselassie | Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy[END_REF] Moreover, antagonism of mitochondrial fission slowed disease progression in a mouse model of amyotrophic lateral sclerosis and abrogated inflammation associated with septic encephalopathy. [START_REF] Joshi | Inhibition of Drp1/Fis1 interaction slows progression of amyotrophic lateral sclerosis[END_REF][START_REF] Haileselassie | Mitochondrial dysfunction mediated through dynamin-related protein 1 (Drp1) propagates impairment in blood brain barrier in septic encephalopathy[END_REF] Further advances in our understanding of fission-mediated inflammatory responses may therefore provide opportunities for targeting inflammation-mediated disease in the future. In summary, LPS promotes acute S635 phosphorylation and sustained S656 dephosphorylation of Drp1, recruitment of Drp1 to mitochondria, mitochondrial fission and Drp1-dependent inflammatory responses in macrophages (Figure 5). Future studies should address the involvement of each of these post-translational modifications in specific macrophage inflammatory responses and the molecular mechanisms involved.

METHODS

Ethics statement

All experiments involving primary human cells were approved by the University of Queensland Medical Research Ethics Committee (approved certificate numbers 2009001051 and 2011000826). Use of primary mouse cells was approved by The University of Queensland Animal Ethics Committee (IMB/123/18, UQDI058/19).

Reagents, animals, cell isolation and culture, quantitative PCR and quantification of mitochondrial DNA copy number and microscopy quantification These details are provided in Supplementary material.

Gene silencing

BMMs were resuspended in media containing HEPES (10 mmol L À1 ; Gibco, Waltham, MA, USA). Indicated small interfering RNAs (Supplementary table 1, 1 lM final), 300 lL cell suspension and media up to a total volume of 400 lL were added to a 0.4-cm cuvette. The cuvette was then subjected to electroporation using a Gene Pulser Xcell Electroporation System (Bio-Rad, Hercules, CA, USA) (240 V, 1000 lF, resistance of ∞). Cells were allowed to recover at room temperature (RT) for 10-15 min and then plated at a concentration of 0.5 9 10 6 cells mL À1 .

Microscopy

BMMs (2 9 10 5 ) or MEF (2.5 9 10 4 ) cells were plated on cover slips in 24-well plates. For mitochondrial staining, after appropriate stimulation, media was replaced and new media containing MitoTracker Deep Red FM (Invitrogen, Waltham, MA, USA; 150 nmol L À1 for BMMs and 200 nmol L À1 for MEF cells) was added for 30 min. Cells were washed three times with phosphatebuffered saline (PBS (Gibco); 5 min each wash) before fixing with 4% paraformaldehyde for 15 min at RT. After fixation, cells were washed three times with PBS and stained with 4 0 ,6-diamidino-2phenylindole (20 ng mL À1 ) for 30 min. For immunostaining with anti-Tom20 and anti-Hsp60 antibodies, after fixing with 4% paraformaldehyde (Sigma, St. Louis, MO, USA), cells were permeabilized using 0.1% Triton X-100 (Sigma) in PBS for 5 min. Next, cells were blocked in 0.5% bovine serum albuminin PBS for 1 h at RT. Cells were then incubated with primary antibodies, rabbit anti-Tom20 or rabbit anti-Hsp60, diluted in 0.5% bovine serum albumin in PBS for 1 h at RT, followed by incubation in a secondary antibody (goat antirabbit Alexa Fluor 488) diluted in 0.5% bovine serum albumin in PBS and 4 0 ,6diamidino-2-phenylindole (20 ng mL À1 ) for 1 h at RT. The coverslips were mounted using mounting media (homemade IMBiol) and cells were imaged using a Zeiss Axiovert 200 Upright Microscope Stand with LSM 710 Meta Confocal Scanner and Spectral detection with 639 magnification (Zeiss, Oberkochen, Germany).

ELISA and nitrite production

BMMs were plated in 96-well plates at 100 000 cells per 100 µL overnight, after which cells were pretreated with mitochondrial fission inhibitors M1 (20 µM) and mdivi1 (10 µM) for 1 h, prior to stimulation with LPS (100 ng mL À1 ) for the indicated time points.

Figure 2 .

 2 Figure 2. LPS-inducible mitochondrial fragmentation requires Drp1. (a-f) The indicated cell populations were stimulated with LPS (100 ng mL À1 ) or left untreated for 6 h, prior to performing indicated analyses. Mitochondrial numbers were determined relative to the unstimulated control in each experiment. (a) BMMs were electroporated with no siRNA or siRNAs targeting either a control gene (Hdac1) or Drp1, incubated for 18 h and then treated with LPS. Cells were stained with MitoTracker (mitochondria, red) and DAPI (nuclear DNA, blue). (b) WT and Drp1 À/À MEF cells were treated with LPS, before being stained with MitoTracker (mitochondria, red) and DAPI (nuclear DNA, blue). Images in a and b are representative of a minimum of three independent experiments. (c, d) Quantification of the relative number of mitochondria in a and b. (c) Mean AE s.e.m. of four independent experiments (calculated using two-way ANOVA with Sid ak's multiple comparisons test) and (d) mean AE s.e.m. of three independent experiments (calculated using two-way ANOVA with Sid ak's multiple comparisons test). (e) BMMs were LPS treated before being stained with MitoTracker (mitochondria, red), DAPI (nuclear DNA, blue) and anti-Drp1 (white). Images are representative of three independent experiments. (f) Relative colocalization of Drp1 with MitoTracker, with data displayed relative to unstimulated control cells (mean AE s.e.m., three independent experiments, one sample t-test). In all panels, *P < 0.05, ***P < 0.001. Scale bars, 5 µm. BMMs, bone marrow-derived macrophages; Con, control; DAPI, 4 0 ,6-diamidino-2-phenylindole; LPS, lipopolysaccharide; MEF, mouse embryonic fibroblast; ns, not significant; siRNA, small interfering RNA; WT, wild type.

Figure 3 .

 3 Figure 3. LPS induces acute Drp1 phosphorylation at S635 and sustained dephosphorylation at S656 in macrophages. (a) Amino acid sequence comparison for the indicated residues of RefSeq human DRP1 variant 1 (isoform 1) and RefSeq mouse Drp1 variant 6 (isoform e), with indicated serine (S) phosphorylation sites on each sequence. (b) BMMs were stimulated with LPS for the indicated time points, after which whole cell extracts were collected and analyzed by western blot for phospho-Drp1 (S635), total Drp1, Tom20 and a-tubulin. Immunoblots are representative of four independent experiments. (c) Quantitative analysis of phospho-Drp1 (S635) relative to total Drp1 levels, with all data plotted relative to the unstimulated control cells (À). Data are combined from four independent experiments [mean AE s.e.m., one-way ANOVA with Dunnett's multiple comparisons test]. (d) Cells were stimulated with LPS for the indicated time points, after which whole cell extracts were collected and analyzed by western blot for phospho-Drp1 (S656), total Drp1 and a-tubulin. Immunoblots are representative of four independent experiments. (e) Quantitative analysis of phospho-Drp1 (S656) relative to total Drp1 levels, with all data plotted relative to the unstimulated control cells (À). Data are combined from four independent experiments (mean AE s.e.m., one-way ANOVA with Dunnett's multiple comparisons test). (f) Mitochondrial numbers (relative to unstimulated control cells) at different time points following LPS stimulation (mean AE s.e.m., four independent experiments, one-way ANOVA with Dunnett's multiple comparisons test). (g) Wild-type and MyD88 À/À BMMs were stimulated with LPS as indicated. Whole cell extracts were prepared, and then analyzed by western blot for phospho-Drp1 (S635), phospho-Drp1 (S656), total Drp1, MyD88 and a-tubulin. The immunoblot is representative of four experiments. In all panels, *P < 0.05. BMMs, bone marrow-derived macrophages; DRP1, dynamin-related protein 1; LPS, lipopolysaccharide; WT, wild type.

Figure 5 .

 5 Figure5. Current model of mechanisms of TLR4-inducible mitochondrial fission and its proinflammatory effects. Upon Tlr4 stimulation, Drp1 is acutely phosphorylated at S635 independently of the adaptor molecule MyD88. By contrast, MyD88 is required for LPS-mediated dephosphorylation of Drp1 at S656 and for mitochondrial fission. Fission enhances the expression of specific proinflammatory genes, for example, Il12b. Drp1, dynamin-related protein 1; IL, interleukin; LPS, lipopolysaccharide; MyD88, myeloid differentiation primary response 88; TLR, Toll-like receptor.

  To induce IL-1b secretion, BMMs were stimulated for 4 h with LPS, followed by treatment with nigericin (10 µM; Sigma, St. Louis, MO, USA) for 4 h. To assess nitric oxide production, BMMs were pretreated with IFNc (5 ng mL À1 ; R&D System, Minneapolis, MN, USA) for 18 h, after which cells were stimulated with LPS for 24 h. Nitrite levels were measured by Griess assay (Promega, Madison, WI, USA) and levels of secreted Il-12p40, Il-6, Il-1b and Tnf were measured by ELISA according to the manufacturer's instructions [TNF and IL-1b kits: BD Biosciences, Franklin Lakes, NJ, USA; IL-6 antibodies: BD Biosciences (Capture: #554400, Detection: #554402); IL-12p40 antibodies (Capture: #551219, Detection: #554476)].ImmunoblottingCells (2 9 10 6 ) were plated in six-well plates and, after appropriate stimulations, they were lysed using radioimmunoprecipitation assay buffer (50 mmol L À1 Tris-HCl, 150 mmol L À1 NaCl, 1 mmol L À1 ethylenediaminetetraacetic acid, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with cOmplete EDTA-free protease inhibitor cocktail (Sigma) and PhosSTOP phosphatase inhibitor tablets (Sigma). Cell lysates (10 µg protein measured by bicinchoninic acid assay; Thermo Fisher Scientific, Waltham, MA, USA) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on to nitrocellulose membranes (Bio-Rad). Membranes were probed with antibodies (Supplementary table 2) and then developed by chemiluminescence using Clarity ECL (Bio-Rad).Experimental data from three or more repeat experiments were combined (taking the mean of technical replicates from each experiment) and GraphPad Prism 8 (San Diego, CA, USA) was used for specific statistical analyses, as indicated in individual figure legends. Data with n ≥ 3 are represented as mean + s.e.m., where n represents number of experiments. Statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) was determined using an unpaired t-test, one sample t-test, one-way or two-way ANOVA, depending on the type of data.

	Statistics

Immunology & Cell Biology 2020; 98: 528-539 www.wileyonlinelibrary.com/journal/icb

14401711, 2020, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/imcb.12363 by Inrae -Dipso, Wiley Online Library on [12/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

ACKNOWLEDGMENTS

This work was supported by a National Health and Medical Research Council of Australia (NHMRC) project grant to MJS and DPF (APP1125316), an NHMRC project grant to JCSJ (APP1160106), as well as a Rebecca Cooper Foundation Research Grant to RK (PG2018095). MJS is supported by an NHMRC Senior Research Fellowship (APP1107914), DPF is supported by an NHMRC Senior Principal Research Fellowship (APP1117017) and the Australian Research Council Centre of Excellence in Advanced Molecular Imaging (CE140100011), RK was supported by an Australian Research Council Discovery Early Career Researcher Award Fellowship (DE1310470), KS is supported by an NHMRC Career Development Fellowship (APP1141131) and JdB was supported by an NL K.F. Hein Fonds Grant (1652430). We acknowledge the support of the Institute for Molecular Bioscience Dynamic Imaging Facility for Cancer Biology at The University of Queensland, established with the support of the Australian Cancer Research Foundation. We also thank the Biological Research Facility at the Translational Research Institute, Dr Nicholas Condon (Institute for Molecular Bioscience, The University of Queensland) for assistance with IMARIS, Dr Alina Zamoshnikova (The University of Queensland Diamantina Institute) for technical support, the Australian Red Cross Blood Service for providing buffy coats for the isolation of human monocytes and Professor Mike Ryan (Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia) for providing Drp1 À/À and wild-type control MEF cells. The funders had no role in study design, data collection and interpretation, or the decision to submit this work for publication.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of the article.


Marine Barral

Hugo Rabier

Anne Termoz

Hassan Serrier

Julie Haesebaert

Laurent Derex

Norbert Nighoghossian

Anne-Marie Schott

Marie Viprey

à la diffusion de documents scientifiques de niveau

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

Patients' productivity losses and informal care cost related to ischemic stroke in Rhone area, France

• Cross-sectional survey, performed along with the STROKE69 prospective population-based cohort study on all IS patients admitted to any emergency department or stroke unit of the Rhône area between November 2015 and June 2016

• At 12 months post-stroke, 2 questionnaires were mailed: one to IS patients and one to their main informal caregivers

• PL were valued with the Human Capital Approach and the Proxy Good Method (PGM).

• IC was valued according the PGM.

Patients' productivity losses and informal care cost related to ischemic stroke in Rhone area, France

Marine Barral1 , Hugo Rabier • First study to value PL including presenteeism and leisure time associated to stroke • PL and IC must be integrated more largely in economic evaluations as it represents a significant cost for society • Economic burden of stroke on society estimated at €64 billion in Europe (1) and €7.7 billion in France (2)

METHODS

RESULTS

Informal

•

  care provided by main informal caregivers to IS patients according to mRS level: care time and associated costs • 222 respondents and analysed (response rate = 42%) • 58% men, mean age = 68.4 yo; 74% retired or unemployed; 86% with a mRs 3-5 at 3 months, 54% received IC Patient's productivity losses during the 1-year post-stroke (€ (SD))

  Abbreviations: mRS = modified Rankin Scale. a Data were available for 51/108 patients. Main reasons for missing data were the absence of estimation of care time or date of discharge from hospital. b mRS at 3 months was missing for 5 patients.

1,2 , Anne Termoz 1,2 , Hassan Serrier 1,2 , Julie Haesebaert 1,2 , Laurent Derex 2,3 , Norbert Nighoghossian 3 , Anne-Marie Schott 1,2 , Marie Viprey 1,2 Financial

support This work was supported by the RHU MARVELOUS (ANR-16-RHUS-0009) of l'Université Claude Bernard Lyon 1 (UCBL), within the program "Investissements d'Avenir" operated by the French National Research Agency (ANR). a 60 individuals were not able to provide a WTP value; b 8 individuals refused to provide a WTP value.

Table 1 .Table 2 .

 12

	Conclusion

Table 2 )

 2 • Concerning the 57 working patients before stroke -Mean PL = €22,459 (including 7% of presenteeism) -35/57 returned to work with a mean delay of 97 days Abbreviations: A= Absenteeism; P= Presenteeism. a Absenteeism concerned all the patients with a professional activity (n=57). b Only 9/57 patients declared being concerned by presenteeism. c Productivity losses concerned 163/165 retired or unemployed patients, as 2/165 were missing data.

	10,069 (5,954)	2,389 (3,843)	7,589 (12,305)

Hospices Civils de Lyon, Pôle Santé Publique, Lyon, France;

University of Lyon 1, University Claude Bernard of Lyon, University of Saint-Etienne, HESPER EA 7425, F-69008 Lyon, F-42023 Saint-Etienne, France;

Hospices Civils de Lyon, Comprehensive Stroke Center, Hôpital Pierre Wertheimer, Bron, France Contact Information : Marine Barral, marine.barral@chu-lyon.fr


Emmanuelle Tabet

Le

est destinée au dépôt

Le Journal d'Henry Bauchau : écriture diaristique et prière Si tu peux/ prier/ demande une âme vide/ attentive/ et ne présumant pas de ses forces. / Tu sens/ et si c'est voir, tu vois/ tes branches suivre la courbe / inespérée du vent. 1 . Bauchau, poète, romancier et psycho-thérapeute, commence à faire publier ses journaux en 1992, à l'âge de 79 ans. Il publie alors, jusqu'à son décès en 2012, une bonne part des journaux qu'il tenait scrupuleusement depuis la fin des années 1930. Les premiers cahiers suivent de près l'engendrement de l'oeuvre romanesque -Jour après Jour retrace les années d'écriture d'OEdipe sur la route, le Journal d'Antigone reflète la création d'Antigone, La Grande Muraille évoque la composition de La Déchirure -alors que les derniers tendent de plus en plus vers le journal poétique et vers des formes de contemplations méditatives, parfois épiphaniques. Mais tout au long de leur rédaction la prière y occupe une place essentielle : le diariste dit prier chaque jour en une pratique s'accompagnant souvent d' « exercices du matin », selon le titre donné à l'un de ses recueils de poèmes 2 . La prière est pour ce par quoi « quelque chose en moi respire 3 », une « respiration naturelle de l'être 4 » -et c'est aussi la définition qu'il donne de l'écriture, qui est pour lui « de l'ordre de la respiration, de la marche, de la nourriture 5 ». Pendant vingt-cinq ans il se récite en particulier tous les jours l'hymne à l'amour de saint Paul dans la Première Épitre aux Corinthiens 6 . Dans ses dernières années il n'a plus la possibilité d'accompagner de gestes sa prière. Il se concentre alors sur la récitation des Béatitudes, et en particulier « Heureux les doux, ils auront la terre en partage », dont il tire une leçon d'abandon et de dépossession :

La douceur, le continent inconnu de la bonté, comme l'appelait Apollinaire, nous ouvre le monde non pour nous l'approprier, mais pour le voir, bénéficier de ses dons, être en lui pleinement. Peut-être lui appartenir plutôt que le posséder 7 .

Pour le diariste qui commente les Béatitudes à travers Apollinaire et qui affirme avoir « tenté de vivre en poésie ce qui [lui] semblait valable de l'oeuvre de beauté et de prière du Thoronet 8 », la prière est intimement liée tant à l'inspiration poétique qu'à l'écriture diaristique. Prier c'est pour lui se laisser traverser par le « souffle du vent », c'est se rendre réceptif à cette dictée intérieure qui est aussi celle du poète, c'est, comme dans l'écriture, « laisser monter en soi ce qui vient d'une source inconnue 9 ». À la suite de Michaux, Bauchau ne cesse de rappeler que « poète n'est pas maître chez lui 10 » : l'épiphanie du poème est une forme d'accueil de ce qui se passe en soi -accueil qui suppose une certaine disposition intérieure faite à la fois de passivité et d'écoute 11 . Mais il aspire aussi à inscrire cette inspiration spirituelle dans une écriture quotidienne, répétée jour après jour comme 1 Poème inédit « Si tu peux », Revue internationale Henry Bauchau, n°1, 2008, p.13.

2 Henry Bauchau, Exercices du Matin, Paris, Actes Sud, 1999. 3 Henry Bauchau, 26 mars 1985, Jour après Jour (JJ), Bruxelles, Les éperonniers,1992, p. 76. 4 Voir Un arbre de mots, entretien avec Indira de Bie, Clichy, Éditions de Corlevour, 2007, p. 20 : « Répond à la question : "Quelle définition donneriez-vous de la prière ?" : "La prière, pour moi, est une respiration. Une respiration naturelle de l'être. Je n'imagine pas que l'on puisse durablement, au cours d'une vie, se confier à ses propres forces, à moins d'être dans une illusion profonde. le sont les gestes de la prière. L'écriture diaristique fondée sur une éthique de régularité rencontre alors l'aspiration de l'orant à une ténacité qui seule permet de maintenir l'espérance : Écrire et prier avec ténacité. Comme fait la nature en somme dans son perpétuel écoulement, sa diversité, sa dépendance au soleil, sa confiance incessante en lui 12 .

Tout au long du journal, Bauchau réaffirme dès lors le lien indissoluble entre une écriture où « se poursuit vraiment [s]on interrogation spirituelle » et la prière sans laquelle il serait « vite à court d'inspiration et très vite de respiration 13 ». Aussi son journal contient-il une réflexion intense à la fois sur la dimension précaire de l'écriture poétique et sur les liens qui se tissent entre l'exercice spirituel quotidien qu'est l'écriture journalière et la prière.

La prière est à la fois un texte et un acte, qui mêle mots, gestes, intentions et convictions [START_REF] Patrick | La Parole et la Prière au Moyen-Âge[END_REF] .

Reflet des élans de l'âme, elle suppose une prééminence des dispositions intérieures sur le contenu de la prière : l'esprit, selon les recommandations de saint Augustin, doit être « en étroite connivence avec les paroles prononcées 15 ». Cette fidélité du texte à une forme d'authenticité de soi, cette « consonance » de l'écriture avec la vérité de l'être est, comme la prière, constitutive de la définition de l'écriture diariste, comme elle l'est de la démarche analytique. En ce sens Bauchau voit une analogie entre l'analyse et la prière : l'analyse est à ses yeux, comme la prière, un état d'écoute profonde, un « état de patience » -une patience qui « redonne confiance à l'inconscient et lui ouvre l'espace où se déployer 16 ». Prière, écriture et rêve s'entremêlent dès lors dans une même relation profonde à l'inconscient et à ce que Bauchau perçoit comme une dictée intérieure :

Il faut passer par là, cette nuit dans un moment de demi-éveil et de prière j'ai senti une grande douceur, inconnue jusqu'ici, pénétrer cette prière que je retrouvais sur mes lèvres au sortir du sommeil 17 .

Du « bonheur calme » qui est celui du semi-éveil émanent des mots qui prennent, dans cet instant où le poète est encore connecté à l'inconscient, une résonance particulière proche de celle de la prière 18 . De même, le poème est décrit comme un souffle qui le traverse « pour se charger de mots 19 ». Et du reste beaucoup de ses poèmes sont, selon l'expression du Dernier Journal, « des prières inconscientes 20 ».

Écrire au jour le jour implique en effet à la fois une variété propre à la diversité même de l'existence, à son ondoyante dispersion, et un principe de répétition, de monotonie, où la succession des jours ne peut se dire qu'à travers une série lancinante de reprises. Comme dans la prière, le renouvellement permanent de l'écriture est une promesse de transformation intérieure et de transfiguration de la répétition en régénération. Aussi les derniers volumes du journal sont-ils traversés par ce qui apparaît comme un refrain : « Les roses trémières de Montour émeuvent toujours mon coeur : Adhésion totale à ce qui est [START_REF] Bauchau | La question qui pourrait alors se poser est celle du destinataire : l'autodestination du journal est-elle compatible avec le fondement de la prière qui est l'adresse ? « Cui narro haec ?[END_REF] . » Le journal est également traversé par le verset de saint Jean sans cesse répété et commenté : « Le vent souffle où il veut et tu entends sa voix, mais tu ne sais pas d'où il vient ni où il va 22 . » Et bien des notations apparaissent comme des variations autour de la sentence paulinienne que Bauchau désigne comme « une admirable prière » : « Ma grâce te suffit. » Le jour de son quatre-vingt-troisième anniversaire, il note ainsi : Chaque jour, depuis de nombreuses années, je me redis la phrase de saint Paul, lue sur une tombe au cimetière des vignerons aux Epesses : « Ma grâce te suffit. » Cette pensée n'a cessé d'orienter ma vie et mon oeuvre 23 .

Ces refrains sont à la fois le leitmotiv, la basse continue du journal et le lieu d'approfondissements successifs. Ainsi les mots de l'Épitre de saint Paul : « Ma grâce te suffit », si souvent répétés dans ses prières matinales, prennent au fil des jours une résonance nouvelle, comme en témoigne la notation du 25 septembre 1986 : je me suis redit cela si souvent que j'avais oublié la suite dans l'Épître de saint Paul : « Ma puissance donne souvent sa mesure dans la faiblesse » […] Ainsi la faiblesse que je ressens si fort est le lieu même où peut se manifester la puissance divine […]. En somme il s'agit de s'effacer et cependant d'être là, d'être présent, puisque nous sommes l'instrument nécessaire d'une action plus vaste dont nous ne pouvons voir la véritable étendue 24 .

Mais si elle fait très souvent référence à saint Paul, cette prière n'est pas pour autant confessionnelle :

La prière a une place importante dans ma vie, je ne pourrais concevoir mes journées sans elle, mais tout le grand corps de l'Église, si appesanti par l'histoire, par l'esprit doctrinal, par les tabous, n'est plus le corps mystique et libre auquel je voudrais appartenir et auquel, peut-être, j'appartiens 25 .

Ses Exercices du matin n'évoquent les exercices spirituels de Loyola que pour s'en éloigner, pour se prosterner « devant rien » et « sans église [START_REF] Bauchau | Exercices du matin[END_REF] ». Dans le Journal d'Antigone il exprime à maintes reprises à quel point sa prière est une interrogation profondément habitée par le mystère, sans réponse, « une interrogation abrupte, s'élevant parfois dans l'allégresse de la nature et parfois sombre et dévastée 27 ». Sa « prière hésitante, difficile » s'adresse à un « dieu inconnu 28 ». Le temps des appartenances confessionnelles est pour lui révolu mais pas le temps du sacré : « Le temps des dieux -et le temps de Dieu -est terminé. Mais l'essentiel qui était derrière le nom de Dieu ne disparaîtra jamais 29 . » La prière de Bauchau réunit alors les trois voies spirituelles de l'Évangile, du Tao et de la poésie : « Le renoncement, la douce et subtile indifférence au succès, c'est la voie difficile indiquée par le Tao Te King, par l'Évangile et, près de nous, par Mallarmé 30 . » Ainsi la sagesse du vent résonne à la fois de la mystique chrétienne et de l'adhésion bouddhiste ou taoïste à ce qui est. Bauchau lit et relit le Tao-tö-King et il est touché par « la justesse, la solidité, la sobriété de cette pensée 31 » qui lui inspire une philosophie du non-vouloir : « Ne pas vouloir, ne pas vouloir agir, laisser se faire 32 . » le diariste n'écrit pas à Dieu -qui de toute façon est déjà au courant de tout ce qu'il pourrait lui dire -mais auprès de Dieu. En réalité la destination externe par excellence -écrire à Dieu -est aussi impossible que la pure autodestination dans la mesure où, dans les deux cas, le narrateur se trouve dans « l'insoluble situation paradoxale d'écrire ce qu'il sait déjà 34 » ou ce que l'Autre connaît déjà. La prière de Bauchau est alors moins une adresse à Dieu qu'une façon d'écrire auprès de Dieu, dans un état de réceptivité, car écrire c'est pour lui se laisser traverser par ce qui le dépasse :

Ce livre sera peut-être le dernier, je l'ignore […]. Mes forces déclinent régulièrement. Peut-être me permettentelles encore d'écrire ? Ce n'est plus dans mes mains, mais dans une force plus grande, celle de la nature, celle du ciel 35 .

La prière est donc moins un acte de parole qu'un état particulier :

Prière dans l'état de détente où je suis, la prière me vient naturellement aux lèvres indépendamment de toute demande ou de toute foi. Elle semble être un état. Du corps ou du coeur, je ne sais [START_REF] Bauchau | La Grande Muraille (GM)[END_REF] .

Cette prière que Jean-Louis Chrétien définissait comme un « acte de présence à l'invisible 37 » se définit moins par un contenu de paroles que par une disposition particulière faite de réceptivité à l'inspiration qui nous traverse : « Je m'aperçois très souvent, note-t-il, que la prière s'échappe de moi sans que je le veuille 38 . » Cet état de réceptivité auquel dispose la prière est propice à l'expérience épiphanique :

  " » 5 Henry Bauchau, 8 août 1984, JJ, p. 31. 6 Henry Bauchau, 18 avril 2006, Dernier Journal (DJ) Paris, Actes Sud, 2015, p. 76. 7 Ibid., 22 septembre 2007, p. 212. 8 Henry Bauchau, 9 mai 2005, Le Présent d'incertitude (PI), Paris, Actes Sud, 2007, p. 245.

9

Henry Bauchau, 25 novembre 1989, Journal d'Antigone (JA), Paris, Actes Sud, 1999, p. 17. 10 Henri Michaux, « L'avenir de la poésie », 24 septembre 1936, in OEuvres complètes, Paris, Editions de la Pléiade, 1998, t.I, pp.967-970.

11 « En marchant dans la rue, le long de la Bibliothèque Nationale, je sens que quelque chose se prépare à prendre possession de moi…Je n'interviens pas, je laisse faire ce qu'il veut. De loin, je le sens prendre forme. » (Henry Bauchau, 4 novembre 1984, JJ, p. 54)