Dmitri V Krysko

Agnieszka Kaczmarek

Olga Krysko

Liesbeth Heyndrickx

Jerzy Woznicki

Pieter Bogaert

Anje Cauwels

Nozomi Takahashi

Stefan Magez

Claus Bachert

Peter Vandenabeele
email: peter.vandenabeele@dmbr.ugent.be

TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation

Keywords: doxorubicin, TLRs, apoptosis, neutrophils, DAMPs, immunogenic cell death

Introduction

Apoptotic cell death is a tightly regulated physiological form of cell death, and one of its important aspects is targeted elimination of apoptotic cells without induction of inflammation or tissue scarring. This process serves as an integral part of homeostatic mechanisms. Apoptotic cells exert anti-inflammatory effects by triggering the release TGF-β, IL-10, platelet-activating factor and prostaglandin E2 from engulfing cells [START_REF] Fadok | Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF[END_REF][START_REF] Stuart | Inhibitory effects of apoptotic cell ingestion upon endotoxin-driven myeloid dendritic cell maturation[END_REF][START_REF] Voll | Immunosuppressive effects of apoptotic cells[END_REF]. In addition, it has been shown that direct contacts between apoptotic cells and phagocytes contribute to the immunosuppressive effect of apoptotic cells [START_REF] Cvetanovic | Specific recognition of apoptotic cells reveals a ubiquitous and unconventional innate immunity[END_REF][START_REF] Cvetanovic | Innate immune discrimination of apoptotic cells: repression of proinflammatory macrophage transcription is coupled directly to specific recognition[END_REF]. Apoptotic cells are cleared via a 'zipper'-like mechanism of internalization [START_REF] Krysko | Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells[END_REF]. In vivo, apoptotic cells are rapidly sensed and cleared not by neutrophils but preferentially by monocytes, which are attracted to the site of cell death by the release of chemoattractants. Elliott et al. [START_REF] Elliott | Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance[END_REF] reported that supernatant obtained from apoptotic Jurkat cells, killed by UV or anti-Fas, induces recruitment of monocytes in a P 2 Y 2 -dependent manner by releasing nucleotides such as ATP and UTP. In fact, apoptotic cells generate negative signals that could prevent neutrophil recruitment and dampen responsiveness of the innate immune system. Recently, it was shown that apoptotic Burkitt lymphoma cells specifically inhibit neutrophil chemotaxis by secreting lactoferrin, a pleiotropic glycoprotein with anti-inflammatory properties, but they do not affect monocyte chemotaxis [START_REF] Bournazou | Apoptotic human cells inhibit migration of granulocytes via release of lactoferrin[END_REF].

The inhibition of neutrophil recruitment by apoptotic cells represents an effective mechanism for limiting tissue injury and inflammation. It is known that the recruited neutrophils and the release of their proteolytic enzymes contribute to many pathological inflammatory conditions, such as ischemic injury of the heart [START_REF] Romson | Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog[END_REF], lung [START_REF] Hall | The role of leukocyte depletion in reducing injury to the lung after hypothermic ischemia[END_REF] and skeletal muscle [START_REF] Sadasivan | Neutrophil mediated microvascular injury in acute, experimental compartment syndrome[END_REF], as well as to toxic insults to the liver [START_REF] Liu | Neutrophil depletion protects against murine acetaminophen hepatotoxicity[END_REF] and lung [START_REF] Ghio | Reduction of neutrophil influx diminishes lung injury and mortality following phosgene inhalation[END_REF].

It was recently shown that chemotherapeutic agents, such as anthracyclines, can induce an immunogenic form of apoptotic cell death [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF][START_REF] Casares | Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death[END_REF], which stimulates the induction of an adaptive immune response that eradicates tumors. The immunostimulatory properties of these immunogenic apoptotic cells are explained by the action of damage-associated molecular patterns (DAMPs) or cell death-associated molecules (CDAMs) [START_REF] Matzinger | The danger model: a renewed sense of self[END_REF][START_REF] Zitvogel | Decoding cell death signals in inflammation and immunity[END_REF]. DAMPs are mostly intracellular molecules that acquire immunostimulatory activity following their secretion or release by stressed or dying cells.

Thus, immunogenic apoptotic cell death is characterized by surface exposure of calreticulin (CRT) [START_REF] Obeid | Calreticulin exposure dictates the immunogenicity of cancer cell death[END_REF] and release of high mobility group box 1 protein (HMGB1) from the nucleus of dying tumor cells into the extracellular space [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. Interestingly, DAMPs are recognized by host cells expressing a set of receptors known as pattern-recognition receptors (PRR), such as Toll-like receptors (TLRs), the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and RIG-I-like receptors (RLRs) [START_REF] Rubartelli | Inside, outside, upside down: damage-associated molecularpattern molecules (DAMPs) and redox[END_REF][START_REF] Kono | How dying cells alert the immune system to danger[END_REF]. When these receptors are triggered by cells dying in an immunogenic or non-physiological way, they rapidly initiate host defense responses. For example, it has been shown that HMGB1 is required for the induction of a doxorubicin-induced anti-tumor immune response in a TLR-4/MyD88 dependent pathway in mice [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. Several reports address the interaction of the adaptive immune system with immunogenic apoptotic cells, but it is not fully understood how the cells of the innate immune system, such as neutrophils, react to immunogenic apoptotic cells.

Given that doxorubicin induces immunogenic apoptotic cell death and that classical apoptotic cells preferentially stimulate monocyte recruitment, we sought to determine how cells killed by doxorubicin are sensed by the innate immune system. We established an in situ murine model of apoptosis induction by intraperitoneal (i.p.) injection of doxorubicin. Intraperitoneal injection of doxorubicin resulted in the generation of mostly apoptotic monocytes/macrophages and induced an acute inflammatory response in the peritoneal cavity characterized by recruitment of neutrophils and production of IL-6 and MCP-1. This acute inflammatory response was specific to immunogenic chemotherapeutics because a non-immunogenic drug, mitomycin C, also induced cell death in the peritoneal cavity but was incapable of eliciting neutrophil attraction. We found that MyD88 is essential for the doxorubicin-induced acute inflammatory response and that it is required primarily as an adaptor molecule in the TLR-2 and TLR-9 signaling pathways.

Materials and Methods

Mice

For the kinetics and dose response experiments, female C57BL/6 and Balb/c mice (8-10 weeks old) were purchased from Janvier (Bio Services BV, The Netherlands). The following mutant mice were used: Myd88 -/-(Balb/c background); TLR-2 -/-, TLR-4 -/-and TLR-9 -/-(C57BL/6 background); TLR-3 -/-(B6129 background); and caspase-1 -/-(6x back crossed to C57Bl/6). Mutant TRIF Lps2 mice (C57BL/6 background) were generated by random mutagenesis with ENU; these mice have a distal frame shift error in a TRIF protein that impairs its function [START_REF] Hoebe | Identification of Lps2 as a key transducer of MyD88-independent TIR signalling[END_REF]. For experiments with knockout mice, wild type mice of appropriate background were used, and they were bred under the same animal house conditions as the others. Mice were housed in a SPF facility with 12/12h light/dark cycles and received water and food ad libitum. All experimental procedures were approved by the local Ethics Committee of Ghent University-VIB. Although some mutants were of Balb/c and mixed B6129 genetic backgrounds, we found that doxorubicin-induced sterile inflammatory responses were not different in C57BL/6, Balb/c and B6129 mice (data not shown).

Doxorubicin-induced inflammation in vivo

Doxorubicin hydrochloride (Sigma-Aldricht, Bornem, Belgium) was freshly dissolved in sterile LPS-free 0.9% NaCl (Braun-N.V.S.A, Diegem, Belgium) at 5 mM. Aliquots were frozen and used before each experiment to ensure constant quality. Mice were injected i.p. with 10 mg/kg of doxorubicin (or as otherwise indicated) in 0.2 ml of 0.9% NaCl. Equal volumes of 0.9% NaCl were injected as negative controls. At different time points, animals were euthanized by CO 2 exposure, and their peritoneal cavities were washed with 8 ml of sterile PBS. The peritoneal lavages were centrifuged at 450 g for 5 min and supernatants were stored at -20°C until analyzed for cytokines.

Red blood cells were lysed with ACK cell lysis buffer (Lonza Walkersville, Basel, Switzerland) and the number of peritoneal exudate cells (PECs) was counted in a hematocytometer using trypan blue.

The doxorubicin solution used for injection contained less than 42 pg/ml endotoxin as measured by Pyrochrome Chromogenic Endotoxin Test kit (Nodia, Antwerp, Belgium).

To control specificity of doxorubicin-induced acute inflammation, mice were injected i.p. with 3 or 10 mg/kg of mitomycin C in 0.2 ml of 0.9% NaCl. Equal volumes of 0.9% NaCl were injected as negative controls. Six hours after injection, PECs were isolated and phenotyped.

Phenotyping of PECs by May-Grünwald-Giemsa staining of cytospins

For differential cell counts, cytospin preparations were made and stained according to the May-Grünwald-Giemsa method, and 250 cells were counted per mouse. Eosinophils, neutrophils, monocytes/macrophages and lymphocytes were identified under a light microscope according to standard morphological criteria. The number of monocytes/macrophages and neutrophils in the PECs was determined by multiplying the total cell numbers by the percentage of monocytes/macrophages and neutrophils, respectively.

Flow cytometric analysis of PECs phenotypes and cell death

PECs (5 × 10 5 ) were incubated with rat anti-mouse antibody 2.4G2 (BD Pharmingen, Erembodegem, Belgium) for 30 min at 4°C to block FcγRIIB/III receptors. Since doxorubicin has a broad range of auto-fluorescence, we divided each sample and used two different stainings in order to identify monocytes/macrophages and neutrophils, and we also examined phosphatidylserine exposure and plasma membrane permeability in both types of cells. In order to quantify monocytes/macrophages, the PECs were stained with anti-mouse antibodies F4/80-APC (clone BM8, eBioscience), CD11b-APC-Cy7 (clone M1/70, BD Pharmingen) and Annexin-V-Alexa Fluor 488 (AnV, Invitrogen, Merelbeke, Belgium). To identify neutrophils, the PECs were stained with anti-mouse antibodies Ly-6G-APC (clone 1A8, BD Pharmingen), CD11b-APC-Cy7 (clone M1/70, BD Pharmingen) and Ann-V-Alexa Fluor 488 (Invitrogen). All the stainings were done for 30 min at 4°C in annexin binding buffer (10 mM HEPES-NaOH, 1 mM MgCl 2 , 2.5 mM CaCl 2 , 5 mM KCl, 150 mM NaCl, pH 7.4). Just before flow cytometry analysis on BD LSR-II (BD Biosciences), 1.25 nM of Sytox Blue dead cell stain was added (Invitrogen). Data were acquired and analyzed by BD FACSDiva software (BD Biosciences). Monocytes/macrophages and neutrophil numbers in the PECs were determined by multiplying the total cell numbers by the percentage of CD11b + F4/80 + and CD11b + Ly6G + cells, respectively. For all kinetics experiments, the PECs were stained only with 1.25 nM of Sytox Red dead cell stain (Invitrogen) and analyzed on FACSCalibur (BD Biosciences). Data were acquired and analyzed by CellQuest software (BD Biosciences). Since Sytox Red and Sytox Blue stain cells with permeabilized plasma membrane in the same way, we use the term "Sytox" for both stainings.

Caspase-3/7 enzymatic activity by fluorometry

The fluorogenic substrate assay for caspase-3/7 activity was carried out as described previously [START_REF] Krysko | Apoptosis and necrosis: detection, discrimination and phagocytosis[END_REF][START_REF] Krysko | Methods for distinguishing apoptotic from necrotic cells and measuring their clearance[END_REF]. Briefly, PECs were washed in cold PBS and lysed in 150 µl of caspase lysis buffer (1% NP-40, 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2 , 1 mM PMSF, 0.3 mM aprotinin, and 1 mM leupeptin) supplemented with 1 mM oxidized glutathione to block the catalytic cysteine of caspases and to prevent their activation during lysis. Cell debris was removed by centrifugation and DEVDase activity was determined by incubating 30 µl (25 ng protein) of the soluble fraction with 20 µM of Ac-DEVD-amc in 150 µl of cell-free system buffer containing 220 mM mannitol, 68 mM sucrose, 2 mM MgCl 2 , 2 mM NaCl, 2.5 mM KH 2 PO 4 , 0.5 mM EDTA, 0.5 mM sodium pyruvate, 0.5 mM L-glutamine, 10 mM HEPES-NaOH (pH 7.4), and 10 mM dithiothreitol. The release of fluorescent 7-amino-4-methylcoumarin was measured for 60 min at 2-min intervals by fluorometry (excitation at 360 nm and emission at 480 nm) (Cytofluor; PerSeptive Biosystems, Cambridge, MA, USA). The maximal rate of increase in fluorescence was calculated ( F/min).

Intraperitoneal injection of MSU

Mice were injected i.p. with 2 mg/mouse of MSU crystals (Enzo Life Sciences, Zandhoven, Belgium) in 200 µl of PBS. Mice injected with equal volumes of PBS served as negative controls.

Six hours after injection, PECs were isolated. They were analyzed for caspase3/7 activity by fluorometry and phenotyped by flow cytometry, as described above.

Cytokine and chemokine analysis

Immunoreactive levels of MCP-1 and IL-6 were measured in peritoneal lavage by using a Cytometric Bead Array (CBA) mouse soluble protein flex set system (BD Biosciences). The samples were prepared according to the manufacturers' instructions and analyzed on a FACSCalibur (BD Biosciences). Data were acquired by CellQuest software (BD Biosciences) and analyzed by FCAP array software (Soft Flow Hungary Ltd.).

Injection of TLR-9 antagonist

Wild type mice were injected i.p. with TLR-9 antagonist (ODN2088, 50 μg) or inactive ODN 2088 (50 μg, Invivogen, Toulouse, France) together with doxorubicin, and six hours later with inactive or active ODN. PECs were obtained 16 hours after doxorubicin injection for cell phenotyping as described previously.

HMGB1 analysis

HMGB1 expression in mouse serum was determined using a specific anti-HMGB1 ELISA (Shino Test Corporation) according to the manufacturer's protocol. Briefly, 100 μl of sample diluent was combined with 10 μl of mouse peritoneal lavage fluid in each well and incubated at 37 °C for 24 h.

Wells were washed five times with wash buffer and incubated for 2 h at 25 °C with 100 μl of PODconjugate solution. Wells were washed five times with wash buffer and incubated for 30 min at room temperature with substrate solution. The reaction was stopped by adding 100 μl of stop solution to each well and the absorbance was read at 450 nm (the background was subtracted by measuring absorbance at 570 nm).

Statistics

Data from multiple experiments are presented as mean ± SEM. Mann Whitney test was used to evaluate the differences between the groups (GraphPad Prism-5 software).

Results

Acute inflammation induced by doxorubicin is associated with apoptosis of monocytes/macrophages

Intraperitoneal injection of doxorubicin resulted in an acute inflammatory response accompanied by the influx of neutrophils and an increase in the levels of IL-6 and MCP-1 in the lavage fluid collected 16 h after doxorubicin injection (Fig. 1). The number of AnnV-positive and Sytoxnegative (AnnV + Sytox -) cells increased in the peritoneum 6 h after i.p. injection of doxorubicin (Fig. 2A), indicating that the majority of PECs were in early stages of apoptosis. To further confirm the type of cell death, caspase activity was determined in the PECs. We found that DEVDase activity (caspase-3/7) was increased in these PECs at 6 h (Fig. 2C), confirming that they were dying by apoptosis. By performing multi-color flow cytometry, we found that the majority of cells which died apoptotically by doxorubicin were mainly monocytes/macrophages with some minor neutrophils (Figs. 2A and2B). Moreover, to exclude the possibility that the observed apoptotic cells were not just regular dying neutrophils, we injected i.p. MSU, which induces strong neutrophil recruitment [START_REF] Chen | MyD88dependent IL-1 receptor signaling is essential for gouty inflammation stimulated by monosodium urate crystals[END_REF]. MSU induced significantly more neutrophil attraction than doxorubicin, but again the number of apoptotic neutrophils in the peritoneal cavity was negligible. Also, no caspase3/7 activity was measured in PECs after i.p. injection of MSU (Fig. 2C). All these data indicate that monocytes/macrophages represent the major cell population that dies by apoptosis after i.p. injection of doxorubicin.

To determine whether neutrophil attraction is induced by the presence of apoptotic cells in the peritoneum, we performed a dose titration experiment in which we investigated the relation between the percent of Sytox + cells and the number of attracted neutrophils as a function of doxorubicin dose. We observed a dose-dependent increase in the percent of Sytox + cells (Figs. 3A and3C).

Moreover, there was a linear correlation between the percent of Sytox + cells and neutrophil influx (Fig. 3B). Notably, the recruitment of neutrophils increased slowly; it was high at 16 h and reached a maximum at 38 h, which is considerably later than the peak of cell death observed at 6 h (Fig. 3D). This time lag between the peak of dead cells and the maximum number of neutrophils implies that neutrophils are attracted after the induction of cell death and that it is associated with cell death.

We used this model to investigate how doxorubicin stimulates acute inflammation in the peritoneum.

Acute inflammation induced by doxorubicin is specific for immunogenic chemotherapeutics

To determine whether an acute inflammatory response, as judged by neutrophil attraction, is specific to the immunogenic anthracyclines, we injected i.p. mitomycin C, which induces a non-immunogenic cell death [START_REF] Obeid | Calreticulin exposure dictates the immunogenicity of cancer cell death[END_REF]. Six hours after i.p. injection of mitomcyin C, there was a significant increase of Sytox + cells (Fig. 4A), but no increase in neutrophil attraction was detected (Fig. 4B). This result indicates that the acute inflammatory response induced by doxorubicin is specific for immunogenic chemotherapeutics.

The role of TLRs in doxorubicin-induced acute inflammation

TLRs recognize signature molecules derived either from pathogens (Pathogen Associated Molecular Patterns, PAMPs) or from dead or damaged cells (DAMPs or CDAMs). When TLRs recognize their ligands, they signal through the TIR adaptor protein MyD88, except for TLR-3, which uses the Toll/IL-1R domain-containing adaptor inducing IFN-α (TRIF). To determine whether TLRs are involved in the inflammation triggered by doxorubicin, we injected doxorubicin i.p. in mice deficient in MyD88 (encoded by MyD88) and mice mutant for TRIF Lps2 mice [START_REF] Hoebe | Identification of Lps2 as a key transducer of MyD88-independent TIR signalling[END_REF], and the acute inflammatory response was evaluated by quantifying the influx of neutrophils. After 16 h, wild-type and TRIF Lps2 mice had abundant neutrophils in their abdominal cavities, but this response was markedly less in MyD88 -/-mice (Figs. 5A and5B). Remarkably, the acute inflammatory response in MyD88-deficient mice was reduced by 22 folds at 16 h (Fig. 5A). Consistent with the reduced neutrophil infiltration in MyD88-deficient mice, the production of IL-6 and MCP-1 was reduced by 12 and 6.5 folds, respectively (Fig. 6B). The percent of Sytox + cells in the MyD88 deficient mice was not different from that in wild type mice (Fig. 3E), indicating that the cell death process itself was not affected.

The requirement for MyD88 in doxorubicin-induced inflammation suggested that TLRs might be involved. Therefore, we injected doxorubicin into mice deficient in various TLRs (TLR-2, TLR-3, TLR-4, or TLR-9). Statistical analysis of the results showed no significant reduction in neutrophil infiltration in mice deficient in TLR-3 or TLR-4 compared with wild-type animals (Figs. 5D and5E). However, the neutrophil response to doxorubicin-induced acute inflammation in TLR-2 and TLR-9 mutant mice was reduced about twofold (Figs 5C and5F), implying that these TLR fulfill an essential role in the recruitment of neutrophils following an apoptotic insult by doxorubicin. However, we do not know whether TLR-1, TLR-5, TLR-6, TLR-7 and TLR-11 (for which we had no knockout mice available) are also involved.

The role of NLRs in doxorubicin-induced acute inflammation

Recently, another family of sensors of PAMPs and DAMPs was identified; this family includes NLRs [START_REF] Martinon | The inflammasomes: guardians of the body[END_REF]. However, although TLRs mediate recognition at the cell surface and at endosomes, NLRs induce innate immune responses upon recognition of PAMPs and DAMPs in the cytosol.

Several NLRs form a caspase-1-activating multiprotein complex, termed the inflammasome, which processes pro-inflammatory cytokines, including IL-1β. It has been shown that dying tumor cells release ATP, which then acts on P 2 X 7 purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex; this allows secretion of interleukin-1β (IL-1β) [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. To investigate whether the inflammasome is important in the doxorubicin-induced neutrophil influx, we injected doxorubicin in caspase-1-deficient mice. The acute inflammatory response in caspase-1-deficient mice was not significantly weaker than in wild-type animals (Fig. 6A). Therefore, NLRs are probably not involved in the induction of the acute inflammatory response to doxorubicin.

TLR-9 antagonist reduces doxorubicin-induced acute inflammation

Having demonstrated that attraction of neutrophils is reduced in TLR-9 -/-mice, we tested whether acute inflammation can be reduced in wild type mice by the administration of a TLR-9 antagonist. Wild type mice were injected i.p. with the active TLR-9 antagonist ODN2088 (50 µg) or the inactive control (50 µg) together with doxorubicin, and 6 h later with inactive or active ODN.

Recruited cells were phenotyped 16 h after doxorubicin injection. The active TLR-9 antagonist significantly reduced the recruitment of neutrophils but the inactive control did not (Fig. 6C). This further confirms the importance of TLR-9 in doxorubicin-induced acute inflammation and also identifies a new therapeutic strategy that might be used to limit the inflammatory side effects of treating peritoneal carcinomatosis by i.p. injection of doxorubicin.

HMGB-1 does not contribute to doxorubicin-induced acute inflammation

Since HMGB1 is one of the DAMPs that could act via TLR-2 [START_REF] Park | Involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein[END_REF][START_REF] Urbonaviciute | Induction of inflammatory and immune responses by HMGB1-nucleosome complexes: implications for the pathogenesis of SLE[END_REF] and TLR-9 (29, 30), which are implied in doxorubicin-induced acute inflammation in our model, we measured the concentration of HMGB1 in the peritoneal lavage. We found that the concentration of HMGB1 in the peritoneal lavage 3, 6 and 16 h after doxorubicin injection was not significantly different from that after injection of vehicle (Fig. 6D).

Discussion

Identifying the signaling pathways involved in the sensing of dead or stressed cells that contribute to the immunomodulatory nature of cell death is critical to our understanding of this fundamental biological process. Apoptosis and necrosis are two major forms of cell death [START_REF] Kroemer | Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009[END_REF]. Classically, apoptosis was described as an immunologically silent and anti-inflammatory form of cell death distinct from necrosis (1-3), whereas necrosis was described as causing stimulation of the immune system [START_REF] Fadok | Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases[END_REF][START_REF] Krysko | Clearance of apoptotic and necrotic cells and its immunological consequences[END_REF].

We established a model for inducing apoptotic cell death in situ by i.p. injection of an anthracycline chemotherapeutic, namely doxorubicin. We show that injection of doxorubicin into the peritoneum induces a sterile inflammatory response characterized by the induction of apoptosis, recruitment of neutrophils, and production of pro-inflammatory mediators, such as IL-6 and MCP-1.

Monocytes/macrophages represent a major target of doxorubicin. Six hours after i.p. injection of doxorubicin, most of the monocytes/macrophages were in early apoptosis (AnV + Sytox). Increased DEVDase activity in the PECs pointed to enhanced activity of caspase-3 and -7, which are crucial executioners of apoptosis [START_REF] Krysko | Methods for distinguishing apoptotic from necrotic cells and measuring their clearance[END_REF]. This agrees with a previous report on doxorubicin-induced apoptosis accompanied by activation of caspase-3 in the human ovarian cancer cell line A2780 [START_REF] Bellarosa | Apoptotic events in a human ovarian cancer cell line exposed to anthracyclines[END_REF] and in the murine colon carcinoma CT26 line [START_REF] Casares | Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death[END_REF]. The kinetic experiments showed that there was a time lag between the appearance of the Sytox + cells (at 6 h) and neutrophil influx (at 16-38 h). Moreover, influx of neutrophils was dose dependent and linearly correlated (r=0.770; p<0.001) with the percentage of Sytox + cells. All these data suggest that the recruitment of neutrophils is associated with apoptotic cells that are killed in situ by doxorubicin. Of note, it has been recently shown that doxorubicin does not have a direct immunostimulatory activity on antigen presenting cells [START_REF] Tanaka | Classification of chemotherapeutic agents based on their differential in vitro effects on dendritic cells[END_REF] thereby excluding a direct effect of doxorubicin on the host cells.

Only some apoptotic stimuli, including anthracyclines, induce an immunogenic form of apoptosis characterized by surface exposure of CRT and release of HMGB1 [START_REF] Obeid | Calreticulin exposure dictates the immunogenicity of cancer cell death[END_REF]. When immunogenic apoptotic cancer cells are implanted into immunocompetent mice, they can convert the 'classical' silent nature of apoptosis into an 'immunogenically active' form of apoptosis. To determine whether only immunogenic chemotherapeutics induce the acute inflammatory response induced by doxorubicin, we injected i.p. mitomycin C, which induces a non-immunogenic form of apoptosis. Indeed, we found that this treatment caused substantial cell death but did not induce neutrophil attraction. These data indicate that the acute inflammatory response induced by doxorubicin is specific for immunogenic chemotherapeutics.

"Classical" apoptotic cells stimulated with traditional apoptotic stimuli, such as anti-Fas antibodies or UV, are sensed by monocytes, which are then recruited in a P 2 Y 2 -receptor dependent manner due to the release of ATP and UTP [START_REF] Elliott | Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance[END_REF]. Apoptotic cells secrete signals, such as lactoferrin, that inhibit the migration of neutrophils but not of monocytes [START_REF] Bournazou | Apoptotic human cells inhibit migration of granulocytes via release of lactoferrin[END_REF]. In contrast, we show that apoptotic cells killed by doxorubicin induce an acute inflammatory response characterized by recruitment of neutrophils but no significant effect on recruitment of monocytes. Thus, apoptotic cells challenged with doxorubicin behave like necrotic cells in terms of neutrophil recruitment. Indeed, it has been shown in a model of sterile inflammation that i.p. injection of heat-killed necrotic cells induces a strong acute inflammation characterized by recruitment of neutrophils [START_REF] Chen | Identification of a key pathway required for the sterile inflammatory response triggered by dying cells[END_REF]. However, in contrast to our results, Chen et al. showed that the IL-1R-MyD88 pathway is important for neutrophil attraction while TLRs are not. The release of IL-1α from necrotic cells was required for production of CXCL-1 and IL-6 by mesothelial cells, which was followed by neutrophil recruitment [START_REF] Eigenbrod | Cutting edge: critical role for mesothelial cells in necrosis-induced inflammation through the recognition of IL-1 alpha released from dying cells[END_REF]. In another study using a model of bacterial peritonitis induced by cecal ligation and puncture, RNA from necrotic cells could amplify the inflammatory response in a TLR-3-dependent manner [START_REF] Cavassani | TLR3 is an endogenous sensor of tissue necrosis during acute inflammatory events[END_REF].

However, in our study, recruitment of neutrophils was independent of TLR-3, and in contrast to the Cavassani study, it required the functions of TLR-2 and TLR-9. The discrepancies between our results and the work of Chen et al. [START_REF] Chen | Identification of a key pathway required for the sterile inflammatory response triggered by dying cells[END_REF] and Cavassani et al. [START_REF] Cavassani | TLR3 is an endogenous sensor of tissue necrosis during acute inflammatory events[END_REF] could be explained by differences in the form of cell death (apoptosis versus primary necrosis) and death stimuli (doxorubicin versus heat shock or ischemia in combination with bacterial peritonitis). The form of cell death and the nature of the stimulus might determine which pathway is used in sensing dead cells. Some NLRs are involved in the recognition of DAMPs released from dead or stressed cells. This recognition can lead to activation of caspase-1 through the assembly of a cytosolic protein complex known as the 'inflammasome" [START_REF] Mariathasan | Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation[END_REF]. It has been shown that dying tumor cells release ATP, which then acts on P 2 X 7 purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. Therefore, we became interested in the possible involvement of caspase-1 in the doxorubicin induced acute inflammatory response in the peritoneum. However, unexpectedly, the acute inflammatory response in caspase-1 deficient mice was comparable to that in wild type mice. Likewise, the level of IL-1β in the peritoneum was not increased after doxorubicin injection in wild type mice (data not shown).

These results indicate that it is unlikely that NLRs are involved in the doxorubicin induced acute inflammatory response.

The presence of both apoptotic and secondary necrotic cells in our model means that release of some DAMPs could contribute to the sterile inflammatory response induced by doxorubicin. Since TLR-3 was not involved in neutrophil recruitment in our model, it is unlikely that RNA acts as a DAMP. But HSPs, a family of highly conserved chaperone proteins, do act as DAMPs when exposed on the surface of dead or stressed cells and when they are released [START_REF] Garg | Immunogenic cell death, DAMPs and anticancer therapeutics: an emerging amalgamation[END_REF]. HSPs are strong immunostimulants and induce neutrophil recruitment. However, several studies indicate that HSPs are recognized mainly by TLR-4 [START_REF] Asea | HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine[END_REF][START_REF] Zou | Critical role of extracellular heat shock cognate protein 70 in the myocardial inflammatory response and cardiac dysfunction after global ischemia-reperfusion[END_REF][START_REF] Chase | Hsp72 induces inflammation and regulates cytokine production in airway epithelium through a TLR4-and NF-kappaB-dependent mechanism[END_REF][START_REF] Wheeler | Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4[END_REF]. Activation of immune cells by HSPs (and possibly by other DAMPs) via TLR-4 may raise concerns about endotoxin contamination masking the observed immunostimulatory effects. In our model, neutrophil attraction induced by apoptotic cells was independent of TLR-4, which excludes the involvement of HSP60, HSP70 or HSP72. The absence of TLR-4 involvement also excludes the possibility that contaminating LPS-like endotoxins contributed to the doxorubicin-induced inflammation.

HMGB1 is another potential DAMP that could be released from necrotic [START_REF] Scaffidi | Release of chromatin protein HMGB1 by necrotic cells triggers inflammation[END_REF] and secondary necrotic cells [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF][START_REF] Urbonaviciute | Induction of inflammatory and immune responses by HMGB1-nucleosome complexes: implications for the pathogenesis of SLE[END_REF]. HMGB1 acts on several receptors, including TLR-2 [START_REF] Park | Involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein[END_REF][START_REF] Urbonaviciute | Induction of inflammatory and immune responses by HMGB1-nucleosome complexes: implications for the pathogenesis of SLE[END_REF] and TLR-9 [START_REF] Tian | Toll-like receptor 9dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE[END_REF][START_REF] Ivanov | A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA[END_REF]. Both TLR-2 and TLR-9 are expressed in various cell types, including neutrophils, monocytes, T cells, B cells, NK cells and dendritic cells [START_REF] Dasari | Expression of toll-like receptors on B lymphocytes[END_REF]. In our model, the concentration of HMGB1 in the peritoneal lavage 3, 6 and 16 h after doxorubicin injection was not significantly different from that after injection of vehicle. Probably most apoptotic cells are engulfed before going into secondary necrosis which may exclude the possible release of HMGB1 from secondary apoptotic cells in our model system. In contrast, previous work has shown that HMGB1 is released from secondary necrotic (late apoptotic) tumor cells in response to doxorubicin treatment [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. This release was implicated in the establishment of an anti-tumor adaptive immune response in a TLR-4 dependent manner. Here, we show that apoptotic/secondary necrotic cells killed in situ by doxorubicin stimulate neutrophil influx independently of TLR-4.

We also show that the sterile neutrophil inflammation induced by doxorubicin can be blocked by a specific TLR-9 antagonist. Further studies are needed to investigate whether blockade of the TLR-2 and TLR-9 pathways could prevent the sterile inflammation induced by doxorubicin without markedly affecting its anti-proliferative effects.

Together, our findings provide important new insights into how the innate immune system senses apoptotic cells killed by immunogenic chemotherapeutic agents. We showed that the acute inflammatory response induced by doxorubicin is specific to immunogenic chemotherapeutics and is associated with apoptosis of monocytes/macrophages. Apoptotic cells killed in situ by doxorubicin are a potential source of DAMPs, which can stimulate TLR-2 and TLR-9 and induce acute inflammation. Our data clearly demonstrate that the TLR-2/TLR-9-MyD88 signaling pathways play a central role in initiating the acute inflammatory response to apoptotic cells induced by doxorubicin administration. We could not identify the DAMPs involved in this process, and it is possible that new molecules are involved in the induction of the acute inflammatory process induced by apoptotic/secondary necrotic cells killed in situ.

Figure 1 .

 1 Figure 1. Intraperitoneal injection of doxorubicin (10 mg/kg) induces a sterile inflammatory

  Figure 2. Intraperitoneal injection of doxorubicin induces apoptosis of monocytes/macrophages. (A) Identification of macrophages/monocytes (F4/80 + CD11b + , total cells) and (B) neutrophils (CD11b + Ly-6G + , total cells) in combination with phosphatidylserine

Figure 3 .

 3 Figure 3. Sterile inflammatory response induced by doxorubicin is associated with apoptosis.

Figure 4 .

 4 Figure 4. Intraperitoneal injection of mitomycin C (MTC) does not induce neutrophil

Figure 5 .

 5 Figure 5. MyD88, TLR-2 and TLR-9 are required for doxorubicin induced neutrophil

Figure 6 .

 6 Figure 6. Role of caspase-1 in neutrophil recruitment and the effect of a TLR-9 antagonist.

Acknowledgments

We thank Prof. S. Akira (for TLR-4 -/-mice), Prof. B. Beutler (for TRIF Lps2 mice), Prof. G. Lauvau (for Myd88 -/-mice), Prof. R. Beyaert and J. Maelfait (for TLR-3 -/-mice). This work was supported by the Fund for Scientific Research Flanders (FWO-Vlaanderen, 3G072810 to D.V.K.) and by an individual research grant from FWO-Vlaanderen (31507110 to D.V.K.). D.V.K. is a postdoctoral fellow and A.K. is a doctoral fellow, both paid by fellowships from FWO-Vlaanderen. Research in the Vandenabeele unit has been supported by Flanders Institute for Biotechnology (VIB), by European grants (FP6 ApopTrain, MRTN-CT-035624; FP7 EC RTD Integrated Project, Apo-Sys, FP7-200767; Euregional PACT II), Belgian grants (Interuniversity Attraction Poles, IAP 6/18) and Flemish grants (Fonds Wetenschappelijke Onderzoek Vlaanderen, 3G.0218.06), Ghent University grants (MRP, GROUP-ID). P.V. is holder of a Methusalem grant (BOF09/01M00709) from the Flemish Government. We thank Dr. Amin Bredan (DMBR-VIB, Ghent) for editing the manuscript.