Anneleen Spooren
email: anneleen.spooren@ugent.be

Pieter Mestdagh
email: pieter.mestdagh@ugent.

Pieter Rondou
email: pieter.rondou@ugent.be

Krzysztof Kolmus
email: krzysztof.kolmus@ugent.be

Guy Haegeman
email: guy.haegeman@ugent.be

Dr Sarah Gerlo
email: sarah.gerlo@ugent.be

K L Ledeganckstraat

IL-1β potently stabilizes IL-6 mRNA in human astrocytes

Keywords: mRNA stability, IL-6, astrocytes, IL-1β, PKC, p38 ARE, AU-rich element, CNS, central nervous system, COX-2, cyclooxygenase 2, ELAV, embryonic lethal abnormal vision, HuR, human antigen R, IL-6, interleukin-6, IL-1β, interleukin-1β, iso, isoproterenol, MAPK, mitogen-activated protein kinase, miRNA, microRNA, PKC, protein kinase C, TNF, tumor necrosis factor, 3"UTR, 3

Uncontrolled expression of IL-6 in the central nervous system is associated with neurodegenerative pathology and glioma development. Astrocytes are the predominant source of IL-6 in the central nervous system, and they are characteristically susceptible to synergistic IL-6 expression. Combined β-adrenergic and TNF-receptor triggering induces synergistic IL-6 expression in 1321N1 cells via a transcriptional enhancer mechanism. Here, we have investigated the molecular basis of the very potent "super"-synergistic IL-6 expression that is apparent after combined treatment of astrocytes with a β-adrenergic agonist, isoproterenol, and the inflammatory cytokines TNF-α and IL-1β. We found that IL-1β treatment strengthens the IL-6 synergy by inducing a distinct stabilization of IL-6 mRNA. Surprisingly, the mRNAstabilizing effect seems to be dependent on protein kinase C (PKC), but not on the prototypical mRNA-stabilizing kinase p38. Moreover, although the mRNA-binding protein HuR basally stabilizes IL-6 mRNA, the mRNA-stabilizing effect of IL-1β is independent of HuR. Our data using pharmacological inhibitors suggest PKC is an important modulator of IL-6 expression in the central nervous system and this might have therapeutic implications.

A c c e p t e d M a n u s c r i p t

INTRODUCTION

Control of the expression levels of cytokines is essential for retaining cellular homeostasis and the orchestration of inflammation. In the central nervous system (CNS), excessive expression of cytokines, such as interleukin-6 (IL-6), has been associated with the pathogenesis and progression of different CNS affections [START_REF] Trikha | Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: a review of the rationale and clinical evidence[END_REF][START_REF] Munoz-Fernandez | The role of tumour necrosis factor, interleukin 6, interferon-gamma and inducible nitric oxide synthase in the development and pathology of the nervous system[END_REF].

For instance, IL-6 has been demonstrated to play an important role in the development and malignant progression of brain glioma by promoting angiogenesis, cell proliferation and resistance to apoptosis and radiation [START_REF] Weissenberger | IL-6 is required for glioma development in a mouse model[END_REF][START_REF] Saidi | Combined targeting of interleukin-6 and vascular endothelial growth factor potently inhibits glioma growth and invasiveness[END_REF][START_REF] Goswami | Interleukin-6-mediated autocrine growth promotion in human glioblastoma multiforme cell line U87MG[END_REF]. Moreover, a positive correlation between IL-6 gene expression and shortened survival in glioblastoma patients has been shown [START_REF] Tchirkov | IL-6 gene amplification and expression in human glioblastomas[END_REF]. During the last decade, more insight has been gained into the physiological consequences of IL-6 upregulation in gliomas; however, the molecular mechanisms leading to excessive IL-6 expression in astrocytes remain largely unclear.

Apart from being involved in tumor development, IL-6 dysregulation in the CNS has been associated with various neurodegenerative diseases, such as Alzheimer"s disease or

Parkinson"s disease. The accompanying neuro-inflammatory reaction is believed to become an enhancer of neurodegeneration at the moment it escapes the normal control mechanisms to restrict the expression of inflammatory mediators, such as cytokines [START_REF] Wyss-Coray | Inflammation in neurodegenerative disease--a double-edged sword[END_REF].

To guarantee their transient expression, many cytokines contain AU-rich elements (AREs) in their 3" untranslated regions (3"UTR). The AREs allow rapid mRNA degradation by promoting the recruitment of mRNA-destabilizing proteins to the 3"UTR. The IL-6 3"UTR contains six AREs [START_REF] Paschoud | Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element, and the RNA-binding protein AUF1[END_REF]. In several systems, IL-6 has been described to have a short half life, varying from 30 minutes [START_REF] Paschoud | Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element, and the RNA-binding protein AUF1[END_REF][START_REF] Ng | Differential induction of the interleukin-6 gene by tumor necrosis factor and interleukin-1[END_REF][START_REF] Weigert | Upregulation of IL-6 mRNA by IL-6 in skeletal muscle cells: role of IL-6 mRNA stabilization and Ca2+-dependent mechanisms[END_REF] to 50 minutes [START_REF] Elias | IL-1 and tumor necrosis factor synergistically stimulate fibroblast IL-6 production and stabilize IL-6 messenger RNA[END_REF]. Several extracellular stimuli have been described to have a stabilizing effect on IL-6 mRNA, such as IL-1β [START_REF] Ng | Differential induction of the interleukin-6 gene by tumor necrosis factor and interleukin-1[END_REF][START_REF] Miyazawa | Regulation of interleukin-1beta-induced interleukin-6 gene expression in human fibroblast-like synoviocytes by p38 mitogen-activated protein kinase[END_REF]; IL-17 [START_REF] Andoh | Extracellular signalregulated kinases 1 and 2 participate in interleukin-17 plus tumor necrosis factor-alphainduced stabilization of interleukin-6 mRNA in human pancreatic myofibroblasts[END_REF][START_REF] Shimada | IL-6 secretion by human pancreatic periacinar myofibroblasts in response to inflammatory mediators[END_REF];

A c c e p t e d M a n u s c r i p t 4 TNF-α [START_REF] Elias | IL-1 and tumor necrosis factor synergistically stimulate fibroblast IL-6 production and stabilize IL-6 messenger RNA[END_REF] and IL-6 itself [START_REF] Weigert | Upregulation of IL-6 mRNA by IL-6 in skeletal muscle cells: role of IL-6 mRNA stabilization and Ca2+-dependent mechanisms[END_REF]. Relatively little is known about the signaling pathways responsible for IL-6 mRNA stabilization. It has been found by multiple groups that the mitogen-activated protein kinase (MAPK) p38 stabilizes IL-6 mRNA [START_REF] Miyazawa | Regulation of interleukin-1beta-induced interleukin-6 gene expression in human fibroblast-like synoviocytes by p38 mitogen-activated protein kinase[END_REF][START_REF] Zhao | p38alpha stabilizes interleukin-6 mRNA via multiple AU-rich elements[END_REF][START_REF] Patil | p38 MAPK regulates IL-1beta induced IL-6 expression through mRNA stability in osteoblasts[END_REF][START_REF] Winzen | The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism[END_REF][START_REF] Neininger | MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different post-transcriptional levels[END_REF]. Another kinase often associated with mRNA stabilization is protein kinase C (PKC). PKC activation has been implicated in mRNA stabilization of, among others, GAP-43 [START_REF] Pascale | Neuronal ELAV proteins enhance mRNA stability by a PKCalpha-dependent pathway[END_REF][START_REF] Perrone-Bizzozero | Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA[END_REF], COX-2 [START_REF] Doller | Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2[END_REF], IL-1β [START_REF] Gorospe | Tumor necrosis factor increases stability of interleukin-1 mRNA by activating protein kinase C[END_REF] and p21 [START_REF] Park | p53-independent elevation of p21 expression by PMA results from PKC-mediated mRNA stabilization[END_REF]. Moreover, PKC and p38 target several RNA-binding proteins that bind 3"UTRs and stabilize or destabilize the mRNA. More specifically for IL-6, direct binding of both AUF-1 [START_REF] Paschoud | Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element, and the RNA-binding protein AUF1[END_REF] and HuR (also known as ELAV-1 (embryonic lethal, abnormal vision, Drosophila-like 1)) [START_REF] Nabors | HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine-and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs[END_REF][START_REF] Zhou | HIV protease inhibitors increase TNF-alpha and IL-6 expression in macrophages: involvement of the RNA-binding protein HuR[END_REF] has already been described, with the former protein both stabilizing and destabilizing IL-6 mRNA, depending on its expression level, and the latter stabilizing IL-6 mRNA.

Astrocytes are the main source of IL-6 in the CNS [START_REF] Van Wagoner | Interleukin-6 expression and regulation in astrocytes[END_REF]. In light of the pathological consequences of uncontrolled IL-6 expression in the CNS, we aimed to unravel the pathways leading to IL-6 production in a human astrocytoma cell line. The 1321N1 cells used in this study were originally isolated from primary cultures of a cerebral glioma multiforme [START_REF] Foster | Glucocorticoids increase the responsiveness of cells in culture to prostaglandin E1[END_REF], and are often used as an astrocyte model [START_REF] Griffin | Persistent interleukin-1beta signaling causes long term activation of NFkappaB in a promoter-specific manner in human glial cells[END_REF]. We have shown before that combined β-adrenergic and TNF-receptor triggering induces synergistic IL-6 expression in 1321N1 cells via a transcriptional enhancer mechanism, involving the cooperative recruitment of CREB, NF-θB and CBP to the IL-6 promoter [START_REF] Spooren | Cooperation of NFkappaB and CREB to induce synergistic IL-6 expression in astrocytes[END_REF]. Here, we describe that IL-1β can further enhance IL-6 production via a post-transcriptional mechanism involving mRNA stabilization. Surprisingly, this effect does not involve a prototypical p38-or HuR-dependent mechanism and, based on pharmacological inhibitor experiments, seems to be PKC-dependent.

A c c e p t e d M a n u s c r i p t 5

MATERIALS AND METHODS

Reagents

Isoproterenol, GF109203X, Brefeldin A (BFA) and actinomycin D were purchased from Sigma-Aldrich (St. Louis, MO). IL-1β was from Invitrogen (Carlsbad, CA). Anti-P-p65-Ser536, anti-P-ERK, anti-P-p38, anti-P-JNK and anti-P-CREB-Ser133 were from Cell Signaling (Danvers, MA); anti-p65 and anti-PARP were from Santa Cruz (Santa Cruz, CA) and anti-tubulin was from Sigma-Aldrich (St. Louis, MO). siHuR SMART pool (M-003773-04) and siControl (D-001210-05) were from Dharmacon (Lafayette, CO). SB203580 was from Alexis Biochemicals (Buttler Pike, PA), Ro31-8220 was from Calbiochem (Gibbstown, MJ). Recombinant human tumor necrosis factor-α (TNF-α) was obtained from the Department of Molecular Biology of Ghent University (DMBR, Ghent, Belgium) (with specific activity of 3.37 10 5 U/μg).

Cell culture

The human astrocytoma cell line 1321N1 was a kind gift from Prof. Dr. Müller (University of Bonn). 1321N1 cells were maintained in Dulbecco"s modified Eagle"s medium, supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin (all from Invitrogen, Carlsbad, CA). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO 2 . All cells were passaged using 0.05% (w/v) of trypsin in 0.4% (w/v) EDTA. Cells were starved overnight prior to inductions in DMEM supplemented with 1% FCS.

Human IL-6 ELISA

A c c e p t e d M a n u s c r i p t 6 Human IL-6 levels were determined using a specific ELISA kit (Biosource, Camarillo, CA) with detection limits of 5 pg/ml, according to manufacturer"s instructions.

RNA isolation and Quantitative real-time PCR

Total RNA was extracted with the acid-guanidinium-thiocyanate-phenol chloroform method using Trizol Reagent (Invitrogen, Carlsbad, CA). Reverse transcription was performed on 0.5 g of total mRNA using MMLV (Promega, Madison, WI). For real time cDNA amplification we used the Biorad SYBR Green Mastermix (Biorad, Hercules, CA) and the following primers: h-IL-6 fw: GAC AGC CAC TCA CCT CTT CA, h-IL-6 rv AGT GCC TCT TTG CTG CTT TC, h-HPRT fw TGA CAC TGG CAA AAC AAT GCA, h-HPRT rv GGT CCT TTT CAC CAG CAA GCT, h-COX-2 fw GCC CTT CCT CCT GTG CC, h-COX-2 rv AAT CAG GAA GCT GCT TTT TAC CTT T, h-IL-8 fw CTC TCT TGG CAG CCT TCC TGA, h-IL-8 rv CAA TAA TTT CTG TGT TGG CGC. Fluorescence was monitored using the BioRad iCycler (BioRad, Hercules, CA). A serial dilution of a cDNA mix standard was used to determine the efficiency of the PCR reaction and to calculate relative mRNA inputs.

Absolute values were normalized to the HPRT reference gene.

Reporter gene assays

The IL-6 promoter luciferase construct containing 1168 bp of the human IL-6 promoter (1168-IL-6-luc) has been described before [START_REF] Vanden Berghe | p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappaB p65 transactivation mediated by tumor necrosis factor[END_REF]. The 3"UTR IL-6 luciferase vector (IL-6

3"UTR-luc, with the IL-6 3"UTR (1-403) cloned in the pGL3 promoter vector, under control A c c e p t e d M a n u s c r i p t of an SV40 promoter) was a kind gift of Prof. Kirkwood (University of Michigan) and has been described before [START_REF] Zhao | p38alpha stabilizes interleukin-6 mRNA via multiple AU-rich elements[END_REF]. FLAG-HuR was a kind gift of Dr. Doller (J.W.Goethe-Universität, Frankfurt am Main) and has been described before [START_REF] Doller | Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA[END_REF]. Cells were seeded in 24well plates (50 000 cells/well) and transfected using the calcium phosphate method with 0.8 g IL-6 construct and 0.2 g neogal constructs per well or with 0.5 g of IL-6 constructs, 0.5 g expression vector plasmid DNA and 0.2 g neogal-construct. 48 hours following transfection, cells were starved overnight and induced for the indicated time period. Total lysates were subsequently incubated with luciferase or galactosidase reagent and luminescence was measured on the Viktor3 system (Perkin Elmer Life Sciences, Boston, MA). Luciferase output was normalized to β-galactosidase values transcribed from the cotransfected galactosidase vector.

Western immunoblotting

Total cellular extracts were made in SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.5% β-mercapto-ethanol). Equal amounts of total lysates from each condition were resolved by 10% SDS-PAGE, transferred onto nitrocellulose membranes and analysed by Western blotting. Chemiluminescent detection was performed using fluorophore-coupled secondary antibodies (Rockland, Gilbertsville, PA) in combination with the Odyssey Imaging System (Licor, Lincoln, NA). Quantification was performed using ImageJ software and based on 3 independent experiments. Tubulin was used as a loading control.

Nuclear and cytosolic extracts were obtained as described earlier [START_REF] Plaisance | Recombination signal sequence binding protein Jkappa is constitutively bound to the NF-kappaB site of the interleukin-6 promoter and acts as a negative regulatory factor[END_REF]. Briefly, cells were lysed in B1 buffer (10 mM HEPES pH 7.5, 10 mM KCl, 1 mM MgCl 2 , 5 % glycerol, 0,5 mM EDTA pH 7.5, 0.1 mM EGTA pH 7.5, 0.5 mM DTT, protease inhibitors and 0.65 % (v/v) A c c e p t e d M a n u s c r i p t 8 NP40). Nuclei were pulled down by centrifugation at 800 rpm for 15 min and nuclear pellets were lysed in B2 buffer by shaking for 15 min at 4°C (20 mM HEPES pH 7.5, 1 % NP40, 1 mM MgCl 2 , 400 mM NaCl, 10 mM KCl, 20 % glycerol, 0.5 mM EDTA pH 7.5, 0.1 mM EGTA pH 7.5, 0.5 mM DTT and protease inhibitors). Lysates were subsequently analyzed by Western blotting. Fractionation was verified using tubulin as a cytoplasmic and PARP as a nuclear control.

siRNA silencing of HuR

For siRNA experiments, cells were seeded in 6-well plates (200 000 cells/well) and transfected using the calcium phosphate method with a final concentration of 40 nM siGENOME SMART pools. siControl transfected samples were used to assess for aspecific effects. 48 hours after transfection and after overnight starvation, cells were induced for the indicated time periods. Subsequently, cells were lysed in SDS sample buffer or RNA was isolated, as described in section 2.4 and 2.6.

Immunofluorescent PKC translocation assay

Immunofluorescence assay of PKC translocation was adjusted from [START_REF] Saha | MAPK p38 regulates transcriptional activity of NF-kappaB in primary human astrocytes via acetylation of p65[END_REF]. Briefly, cells were starved overnight, followed by induction for the indicated time periods. Cells were fixed and permeabalized in ice cold methanol for 5 min at -20°C. Next, samples were blocked for 1 hour at room temperature with PBS 3% BSA and then washed 3 times with PBST (PBS with 0.1 % Tween). Nuclei were coloured with DAPI and the coverslips were mounted using

Actinomycin D assays

For actinomycin D assays, cells were seeded in 6-well plates (250 000 cells/well). After overnight starvation, cells were preinduced with the indicated stimuli and/or inhibitors.

Subsequently media were aspirated and replaced by starvation medium containing 5 μg/ml of actinomycin D to block transcription. After incubation for the indicated time periods with actinomycin D, RNA was isolated, reverse transcription was performed and cDNA was amplified using qPCR with SYBR Green and specific primers for IL-6. Absolute mRNA values were normalized to a housekeeping gene (HPRT) and subsequently recalculated as percentages of mRNA values after preinduction with the respective inductantia without actinomycin D treatment (0 min, 100%).

Analysis of let-7a expression levels

Let-7a expression levels were determined using a specific Taqman microRNA assay (Applied Biosystems, Carlsbad, CA) according to manufacturer"s instructions. Briefly, 1321N1 cells were seeded in 6-well plates (250 000 cells/well), starved overnight and induced for 6 hours. RNA was isolated as described under 2.4, and 100 ng of non-denatured RNA was used for RT-PCR using specific stem-loop primers. cDNA was subjected to qPCR using specific Taqman probes according to manufacturer"s instructions. For normalization, the following small RNA controls were used: RNU6B, RNU44, RNU58B. Stability of these small RNA A c c e p t e d M a n u s c r i p t controls was assessed using the geNorm algorithm implemented in the qbasePLUS Software package (Biogazelle, Ghent, Belgium) [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF].

Statistical Analysis

Results are represented as mean values ± standard error of mean (SEM). Statistical analysis was performed using one-way ANOVA, followed by Bonferroni"s multiple comparison test, except in figure 2i, 4d and 6b where a Student"s t-test was used. Both tests were performed using Graphpad Prism 4 software (Graphpad Software Inc., San Diego, CA). Results were considered as significant when P < 0.05.

IL-1β potently enhances the isoproterenol/TNF-α-induced IL-6 synergy

We have shown before that combined β-adrenergic and TNF receptor triggering elicits synergistic IL-6 expression in human astrocytoma 1321N1 cells at protein, mRNA and promoter level [START_REF] Spooren | Cooperation of NFkappaB and CREB to induce synergistic IL-6 expression in astrocytes[END_REF]. Here we show that addition of the pro-inflammatory cytokine IL-1β to the isoproterenol and TNF-α treatment protocol substantially strengthened this synergistic IL-6 production, inducing a "super"-synergy. This synergy is prominently present at the IL-6 protein level, as measured by ELISA (Fig. 1a), as well as at the mRNA level, as measured by RT-qPCR (Fig. 1b). Analysis of the ratio of IL-6 mRNA after triple (iso+TNF+IL) versus double (iso+TNF) induction indicates a 10.9  1.32 fold enhancement.

IL-1β does not enhance isoproterenol/TNF-α-induced IL-6 promoter activation

The prototypical signaling cascades activated by isoproterenol, TNF-α and IL-1β were screened to investigate if a) there was synergistic activation of one of the downstream kinases or transcription factors and b) if there were factors that were exclusively activated by IL-1β, thereby explaining the synergy. The MAP kinases p38, JNK and ERK1/2, previously shown to be potently activated by TNF-α in 1321N1 cells [START_REF] Spooren | Cooperation of NFkappaB and CREB to induce synergistic IL-6 expression in astrocytes[END_REF], were also activated by IL-1β (Fig. 2a, b, c andf). However, triple induction did not lead to synergistic activation of any of the MAP kinases compared to double induction. Additionally, combined isoproterenol and TNF-α treatment activated CREB [START_REF] Spooren | Cooperation of NFkappaB and CREB to induce synergistic IL-6 expression in astrocytes[END_REF], and IL-1β also induced minor CREB phosphorylation, but A c c e p t e d M a n u s c r i p t again triple induction did not induce synergistic CREB activation as compared to double induction (Fig. 2d andf). Lastly, NF-B activation, measured by translocation (Fig. 2g) and Serine 536 phosphorylation of p65 (Fig. 2e andf), was induced by both iso+TNF and IL-1β.

Again triple induction did not lead to synergistic NF-B activation compared to double induction at 30 minutes. Investigation of the main signaling cascades activated by isoproterenol, TNF-α and IL-1β, thus showed that IL-1β did not activate any "extra" signaling cascades, i.e. that were not already activated by isoproterenol or TNF-α, nor did IL-1β induce synergistic activation of any of the investigated factors. In line with this, we found that, at the promoter level, as measured by a reporter gene assay, triple induction did not significantly strengthen promoter activation, as compared to double induction (Fig. 2h). The ratio of the triple versus double induction was reduced to 1.3  0.28 at the promoter level (Fig. 2i). This ratio does not differ significantly from 1 (P = 0.0959), which indicates that the synergy has disappeared from mRNA to promoter level.

IL-1β stabilizes IL-6 mRNA.

Because the strengthening effect of IL-1β was not conserved at the promoter level, a potential explanation for the synergy could be the stabilization of IL-6 mRNA by IL-1β. It has been shown that IL-1β stabilizes multiple mRNA species, including IL-6, in a variety of cell types [START_REF] Ng | Differential induction of the interleukin-6 gene by tumor necrosis factor and interleukin-1[END_REF][START_REF] Miyazawa | Regulation of interleukin-1beta-induced interleukin-6 gene expression in human fibroblast-like synoviocytes by p38 mitogen-activated protein kinase[END_REF][START_REF] Shimada | IL-6 secretion by human pancreatic periacinar myofibroblasts in response to inflammatory mediators[END_REF][START_REF] Patil | p38 MAPK regulates IL-1beta induced IL-6 expression through mRNA stability in osteoblasts[END_REF][START_REF] Kirkwood | Chemically modified tetracyclines selectively inhibit IL-6 expression in osteoblasts by decreasing mRNA stability[END_REF][START_REF] Van Wagoner | Interleukin-6 (IL-6) production by astrocytes: autocrine regulation by IL-6 and the soluble IL-6 receptor[END_REF]. A paradigm commonly used to study effects on mRNA stabilization is the blockade of new transcription by a transcriptional inhibitor, such as actinomycin D, followed by quantification of the mRNA levels at several time points after inhibition of transcription [START_REF] Ross | mRNA stability in mammalian cells[END_REF]. This allows measuring the degradation rate of a certain mRNA species.

When we investigated the degradation rate of IL-6 mRNA after double versus triple Adding IL-1β to the isoproterenol/TNF-α induction clearly stabilized the IL-6 mRNA by blocking virtually all IL-6 mRNA degradation (Fig. 3a). This effect was sustained for up to 6 hours after addition of actinomycin D (Suppl Fig. 1). We also investigated whether IL-1β induces mRNA stabilization on its own, and, although the IL-1β-mediated mRNA induction is relatively small, complicating establishment of the degradation rate, it was clear from our experiments that IL-1β by itself also stabilizes IL-6 mRNA (Suppl Fig. 2). Furthermore, we analyzed the degradation rates of two other genes that are induced by isoproterenol, TNF-α and IL-1β in 1321N1 cells, namely COX-2 and IL-8 [START_REF] Spooren | Cooperation of NFkappaB and CREB to induce synergistic IL-6 expression in astrocytes[END_REF][START_REF] Aloisi | Production of hemolymphopoietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-1 beta and tumor necrosis factor-alpha[END_REF]. COX-2 showed a synergistic mRNA induction and an mRNA degradation pattern that was very comparable to the one observed for IL-6 (Fig. 3c andd). Triple induction stabilized COX-2 mRNA completely compared to double induction. IL-8, on the other hand, showed a slower, but progressive mRNA degradation pattern, with no significant stabilization by IL-1β at the earlier time points (60 and 120 min, Suppl Fig. 3).

As the 3"UTR is the main target for mRNA-(de)stabilizing mechanisms, we also studied IL-6 mRNA stability using a reporter gene assay. In this system, the IL-6 3"UTR is cloned behind the luciferase coding sequence, thus reporting 3"UTR-mediated effects on mRNA stability via changes in the luciferase signal. Both the double induction with isoproterenol and TNF-α and the individual induction with IL-1β induced a small, but unsignificant increase in the RLU values after 6, 16 and 24 hours induction (Fig. 3b). Only the triple induction with isoproterenol, TNF-α and IL-1β elicited a significant increase in RLU values after 6, 16 and 24 hours, indicating luciferase mRNA stabilization. Two kinases that have been particularly implicated in mRNA-stabilizing effects are p38 and PKC [START_REF] Dean | The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilisation[END_REF][START_REF] Shim | The control of mRNA stability in response to extracellular stimuli[END_REF]. Importantly, both can be activated by IL-1β and have been convincingly implicated in COX-2 mRNA stabilization [START_REF] Doller | Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2[END_REF][START_REF] Doller | Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA[END_REF][START_REF] Faour | Prostaglandin E(2) regulates the level and stability of cyclooxygenase-2 mRNA through activation of p38 mitogen-activated protein kinase in interleukin-1 beta-treated human synovial fibroblasts[END_REF][START_REF] Ridley | A p38 MAP kinase inhibitor regulates stability of interleukin-1-induced cyclooxygenase-2 mRNA[END_REF][START_REF] Mifflin | Regulation of COX-2 expression in human intestinal myofibroblasts: mechanisms of IL-1-mediated induction[END_REF][START_REF] Subbaramaiah | Regulation of cyclooxgenase-2 mRNA stability by taxanes: evidence for involvement of p38, MAPKAPK-2, and HuR[END_REF][START_REF] Lin | Involvement of protein kinase Cgamma in IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells[END_REF][START_REF] Carpenter | Protein kinase Cdelta activation by interleukin-1beta stabilizes inducible nitric-oxide synthase mRNA in pancreatic beta-cells[END_REF]. Moreover, in multiple studies, IL-1β-induced mRNA stabilization of IL-6 was shown to be crucially dependent on p38 [START_REF] Miyazawa | Regulation of interleukin-1beta-induced interleukin-6 gene expression in human fibroblast-like synoviocytes by p38 mitogen-activated protein kinase[END_REF][START_REF] Patil | p38 MAPK regulates IL-1beta induced IL-6 expression through mRNA stability in osteoblasts[END_REF], whereas dependence on PKC has not yet been described. Therefore, we used pharmacological inhibitors of p38 and PKC to investigate if they are involved in the mRNA-stabilizing effect of IL-1β in our experimental system. When we blocked p38 with SB203580, IL-1β was still able to stabilize IL-6 mRNA in the actinomycin D assay (Fig. 4a), indicating that p38 activation is not necessary for IL-6 mRNA stabilization. The effectiveness of SB203580 was demonstrated by the complete inhibition of p38-mediated MSK phosphorylation after treatment with the inhibitor (Fig. 4b). On the other hand, when we blocked PKC with GF109203X, we observed complete inhibition of the stabilizing effect of IL-1β (Fig. 4a). This suggests that IL-1β stabilizes IL-6 mRNA via PKC. To further substantiate the finding that PKC was involved, we used a second PKC inhibitor, namely Ro31-8220. Again we observed complete inhibition of the IL-1β-mediated IL-6 mRNA stabilization (Fig. 4c), whereas parallel inhibition by SB203580 had no effect. Additional evidence for the involvement of PKC came from the ability of PMA, a PKC activator, to mimic IL-1β-induced stabilization of the IL-6 3"UTR reporter gene (Fig. 4d). Moreover, like IL-1β, PMA also induced "super"synergistic IL-6 mRNA production, when added to isoproterenol and TNF-α, imitating the super-synergy induced by IL-1β (Fig. 4e). PKC is a family of kinases composed of 10 isoforms [START_REF] Mellor | The extended protein kinase C superfamily[END_REF]. The family is subdivided into 3 classes, based on their Ca 2+ , diacylglycerol and phospholipid dependency, namely, the classical (α, βI, βII, γ), novel (ε, ζ, δ, ε) and atypical PKCs (η, δ). In order to identify the PKC isoform responsible for the IL-1β-induced IL-6 mRNA stabilization, we set up a large screening experiment using different previously validated PKC-GFP constructs [START_REF] Doller | Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA[END_REF][START_REF] Kajimoto | Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells[END_REF][START_REF] Villalba | Translocation of PKC[theta] in T cells is mediated by a nonconventional, PI3-K-and Vav-dependent pathway, but does not absolutely require phospholipase C[END_REF][START_REF] Raghunath | The catalytic domain limits the translocation of protein kinase C alpha in response to increases in Ca2+ and diacylglycerol[END_REF][START_REF] Babwah | Protein kinase C isoform-specific differences in the spatial-temporal regulation and decoding of metabotropic glutamate receptor1a-stimulated second messenger responses[END_REF][START_REF] Faisal | The scaffold MyD88 acts to couple protein kinase Cepsilon to Toll-like receptors[END_REF].

Identification of the PKC isoform involved

Our aim was to assess which PKC isoform was activated after triple induction, as manifested by PKC translocation to the membrane, and sometimes also to the nucleus. However, at various time points ranging from 1 minute to 60 minutes, we did not observe translocation of any of the 10 isoforms after triple induction, even though PMA, used as a positive control, induced clear membrane translocation of the classical and novel isoforms. An exemplary image of PKC distribution after triple induction or PMA treatment is shown in supplementary figure 4 for the various PKC isoforms at the 30 minute time point. Similar results were obtained at earlier and later time points (1", 5", 10, 15", 30", 60" 6h; data not shown).

The IL-1β-induced IL-6 mRNA stabilization is a late, indirect effect, independent of protein secretion

Since we could block the IL-1β-induced IL-6 mRNA stabilization by PKC inhibitors on the one hand, but did not observe rapid PKC activation after triple induction on the other hand, we hypothesized that the mRNA-stabilizing effect of IL-1β was possibly indirect and delayed.

In line with this, the full-blown IL-1β-mediated mRNA-stabilizing effect was not apparent when we reduced the preinduction time to 1 or 3 hours in our actinomycin D experiments (Fig. 5a). Similarly, IL-1β did not induce COX2 mRNA stabilization after 1 or 3 hours A c c e p t e d M a n u s c r i p t preincubation with isoproterenol, TNF-α and IL-1β combined (Fig. 5b). These data suggest that although IL-1β stabilizes IL-6 mRNA, it does so via triggering a late-onset, possibly indirect, mechanism. To investigate if a secreted factor was responsible for the effect of IL-1β, we blocked secretion of proteins using brefeldin A (BFA). Pretreatment with 5 μg/ml BFA did not inhibit IL-1β-induced IL-6 mRNA stabilization (Fig. 5c). The effectiveness of BFA was demonstrated by visualizing the Golgi network, using the Golgi-marker GM130 (Fig. 5d). Treatment of 1321N1 cells with BFA resulted in the transformation of the discrete perinuclear, vesicular structure to a largely dispersed, dotted structure, reflecting the desintegration of the Golgi network. In addition, we could not mimic the IL-6 mRNAstabilizing effect by using conditioned medium fractions, suggesting the mRNA-stabilizing effect is not dependent on an IL-1β-induced secreted factor (data not shown). We also investigated the role of novel protein synthesis in the stabilizing effect of IL-1β, using the translational elongation inhibitor cycloheximide. However, as described before by several other authors, translational inhibition led to "superinduction" of IL-6 mRNA [START_REF] Kirkwood | Chemically modified tetracyclines selectively inhibit IL-6 expression in osteoblasts by decreasing mRNA stability[END_REF][START_REF] Ross | mRNA stability in mammalian cells[END_REF] (data not shown). This effect has been attributed to the freezing of the translation machinery on the IL-6 mRNA, thereby protecting it from degradation, and thus rules out the possibility of using translational inhibitors to study whether protein synthesis is required for IL-1β induced IL-6 mRNA stabilization [START_REF] Kirkwood | Chemically modified tetracyclines selectively inhibit IL-6 expression in osteoblasts by decreasing mRNA stability[END_REF]. However, when 1321N1 cells are treated with IL-1β in the presence of actinomycin D (instead of the here employed treatment with IL-1β before actinomycin D treatment), IL-6 mRNA is no longer stabilized, indicating the importance of transcription for the mRNA stabilizing effect of IL-1β (data not shown).

IL-1β does not stabilize IL-6 mRNA via a prototypical HuR-dependent pathway

A c c e p t e d M a n u s c r i p t

One of the main mRNA-stabilizing proteins, is the ELAV protein HuR, which upon stimulation with mRNA-stabilizing stimuli translocates from the nucleus to the cytoplasm where it binds and stabilizes the 3"UTRs of several mRNAs [START_REF] Brennan | HuR and mRNA stability[END_REF]. HuR has previously been shown to stabilize IL-6 [START_REF] Nabors | HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine-and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs[END_REF][START_REF] Zhou | HIV protease inhibitors increase TNF-alpha and IL-6 expression in macrophages: involvement of the RNA-binding protein HuR[END_REF] and COX-2 mRNA [START_REF] Doller | Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2[END_REF][START_REF] Doller | Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA[END_REF][START_REF] Doller | Angiotensin II induces renal plasminogen activator inhibitor-1 and cyclooxygenase-2 expression posttranscriptionally via activation of the mRNA-stabilizing factor human-antigen R[END_REF]. Moreover, HuR is a major effector of PKC-mediated mRNA stabilization [START_REF] Doller | Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2[END_REF][START_REF] Doller | Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA[END_REF][START_REF] Doller | Angiotensin II induces renal plasminogen activator inhibitor-1 and cyclooxygenase-2 expression posttranscriptionally via activation of the mRNA-stabilizing factor human-antigen R[END_REF] and can be induced by IL-1β [START_REF] Yeh | RNA-binding protein HuR interacts with thrombomodulin 5'untranslated region and represses internal ribosome entry site-mediated translation under IL-1 beta treatment[END_REF][START_REF] Suswam | IL-1beta induces stabilization of IL-8 mRNA in malignant breast cancer cells via the 3' untranslated region: Involvement of divergent RNA-binding factors HuR, KSRP and TIAR[END_REF]. Therefore, we sought to determine if HuR was involved in the IL-6 mRNA-stabilizing effect of IL-1β.

First, we analyzed the effect of overexpressing HuR in the IL-6 3"UTR luciferase assay.

Although HuR overexpression did clearly enhance the basal luciferase signal (Fig. 6a), there was no effect on the magnitude of the triple induction (Fig. 6b). This suggests that HuR is able to stabilize IL-6 mRNA via its 3"UTR, which corresponds to findings in other studies, but nevertheless the effect of IL-1β does not seem to be mediated via HuR. In line with this, we found that the nuclear/cytoplasmic distribution of endogenous HuR after triple induction at different time points (0-6 hours) did not change (Fig. 6c). In 1321N1 cells, HuR is already present in the cytoplasm in small amounts, possibly due to the fact that they are tumor-derived cells [START_REF] Khabar | Post-transcriptional control during chronic inflammation and cancer: a focus on AU-rich elements[END_REF]. Since HuR has also been shown to stabilize mRNAs in response to IL-1β in the absence of cytoplasmic translocation, but via direct binding to the 3"UTR [START_REF] Suswam | IL-1beta induces stabilization of IL-8 mRNA in malignant breast cancer cells via the 3' untranslated region: Involvement of divergent RNA-binding factors HuR, KSRP and TIAR[END_REF], we next employed an siRNA approach to finally exclude the involvement of HuR. Knockdown of HuR using siRNA did not inhibit IL-1β mediated IL-6 mRNA stabilization (Fig. 6d), even though there was substantial knockdown of HuR protein as assessed by Western blot (Fig. 6e), indicating HuR was not involved in IL-1β-mediated IL-6 mRNA stabilization.

Let-7a microRNA is not involved in the IL-1β-induced IL-6 mRNA stabilization

A c c e p t e d M a n u s c r i p t 18 Because a prototypical p38-or HuR-dependent mechanism did not seem to be involved in IL-6 mRNA stabilization in our experimental system, we additionally investigated whether microRNAs (miRNAs) played a role in IL-6 mRNA stabilization [START_REF] Filipowicz | Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?[END_REF]. miRNAs are particularly abundant in the brain and are suggested to have a pivotal role in several aspects of CNS functioning [START_REF] Kosik | The neuronal microRNA system[END_REF]. Recently, the let-7a miRNA was shown to bind the IL-6 3"UTR [START_REF] Iliopoulos | An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation[END_REF].

The let-7 miRNA family is comprised of 12 members. Importantly, it is was shown that NF-θB activation led to downregulation of let-7a and this subsequently leads to upregulation of IL-6, resulting in a positive feedback loop [START_REF] Iliopoulos | An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation[END_REF]. Since IL-1β has been shown to cause longterm, persistent NF-θB activation in 1321N1 cells [START_REF] Griffin | Persistent interleukin-1beta signaling causes long term activation of NFkappaB in a promoter-specific manner in human glial cells[END_REF], we hypothesized that IL-1β might downregulate let-7a in 1321N1 cells. However, when we analyzed let-7a levels after treatment with isoproterenol, TNF-α and IL-1β, alone or in combinations, we did not observe significant differences in the expression level of this miRNA, indicating that let-7a is not involved in the IL-6 mRNA-stabilizing effect of IL-1β in 1321N1 cells (Fig. 6f).

A c c e p t e d M a n u s c r i p t 4 DISCUSSION

Here, we have reported that IL-1β potently enhances TNF-α/isoproterenol-induced IL-6 expression in astrocytes. Susceptibility to synergy is a hallmark of IL-6 expression by astrocytes [START_REF] Van Wagoner | Interleukin-6 expression and regulation in astrocytes[END_REF]. IL-6 has paradoxical effects in the CNS, combining pro-inflammatory and neurotrophic traits [START_REF] Gadient | Interleukin-6 (IL-6)--a molecule with both beneficial and destructive potentials[END_REF], and dose and timing of IL-6 expression are regarded as crucial determinants in shifting the balance between beneficial and detrimental effects. Therefore, it is important to understand the molecular mechanisms underlying synergistic IL-6 expression in astrocytes.

In 1321N1 cells, IL-1β did not enhance isoproterenol-and TNF-induced IL-6 expression via a transcriptional mechanism, but instead very potently enhanced IL-6 mRNA stability. IL-1β has been previously demonstrated to stabilize multiple mRNA species, such as COX-2 [START_REF] Ridley | A p38 MAP kinase inhibitor regulates stability of interleukin-1-induced cyclooxygenase-2 mRNA[END_REF][START_REF] Mifflin | Regulation of COX-2 expression in human intestinal myofibroblasts: mechanisms of IL-1-mediated induction[END_REF][START_REF] Repovic | Oncostatin M enhances the expression of prostaglandin E2 and cyclooxygenase-2 in astrocytes: synergy with interleukin-1beta, tumor necrosis factoralpha, and bacterial lipopolysaccharide[END_REF][START_REF] Srivastava | IL-1 beta stabilizes COX II mRNA in renal mesangial cells: role of 3'-untranslated region[END_REF][START_REF] Tamura | Interleukin-1beta elevates cyclooxygenase-2 protein level and enzyme activity via increasing its mRNA stability in human endometrial stromal cells: an effect mediated by extracellularly regulated kinases 1 and 2[END_REF], IL-8 [START_REF] Suswam | IL-1beta induces stabilization of IL-8 mRNA in malignant breast cancer cells via the 3' untranslated region: Involvement of divergent RNA-binding factors HuR, KSRP and TIAR[END_REF] and NGF [START_REF] Vige | Mechanism of nerve growth factor mRNA regulation by interleukin-1 and basic fibroblast growth factor in primary cultures of rat astrocytes[END_REF][START_REF] Pshenichkin | Transcriptional and posttranscriptional mechanisms involved in the interleukin-1, steroid, and protein kinase C regulation of nerve growth factor in cortical astrocytes[END_REF]. Importantly, it has already been reported that IL-1β stabilizes IL-6 mRNA in osteoblasts [START_REF] Patil | p38 MAPK regulates IL-1beta induced IL-6 expression through mRNA stability in osteoblasts[END_REF][START_REF] Kirkwood | Chemically modified tetracyclines selectively inhibit IL-6 expression in osteoblasts by decreasing mRNA stability[END_REF], fibroblasts [START_REF] Ng | Differential induction of the interleukin-6 gene by tumor necrosis factor and interleukin-1[END_REF], myofibroblasts [START_REF] Shimada | IL-6 secretion by human pancreatic periacinar myofibroblasts in response to inflammatory mediators[END_REF], astrocytes [START_REF] Van Wagoner | Interleukin-6 (IL-6) production by astrocytes: autocrine regulation by IL-6 and the soluble IL-6 receptor[END_REF] and fibroblast-like synoviocytes [START_REF] Miyazawa | Regulation of interleukin-1beta-induced interleukin-6 gene expression in human fibroblast-like synoviocytes by p38 mitogen-activated protein kinase[END_REF], although relatively little was known about the molecular mechanisms underlying this stabilization. Moreover, other studies found no stabilization of IL-6 mRNA after IL-1β induction, but instead found that TNF-α [START_REF] Elias | IL-1 and tumor necrosis factor synergistically stimulate fibroblast IL-6 production and stabilize IL-6 messenger RNA[END_REF] or cAMP [START_REF] Szabo-Fresnais | Activation of the cAMP pathway synergistically increases IL-1-induced IL-6 gene expression in FRTL-5 thyroid cells: involvement of AP-1 transcription factors[END_REF] were the stabilizing agents in this instance, indicating the cell type specificity of IL-1β-mediated mRNA stabilization.

Two kinases in particular emerge as possible candidates for mediating IL-1β-induced IL-6 mRNA stability, namely p38 and PKC. p38 has been identified as an essential kinase in A c c e p t e d M a n u s c r i p t 20 regulating IL-1β-mediated mRNA stabilization in multiple studies [12, 15-17, 41-44, 56], of which several have specifically investigated IL-1β-mediated COX-2 and IL-6 mRNA stabilization. On the other hand, PKC has been implicated in the stabilization of a variety of other mRNAs such as, p21, GAP-43 and IL-1β itself [START_REF] Pascale | Neuronal ELAV proteins enhance mRNA stability by a PKCalpha-dependent pathway[END_REF][START_REF] Perrone-Bizzozero | Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA[END_REF][START_REF] Gorospe | Tumor necrosis factor increases stability of interleukin-1 mRNA by activating protein kinase C[END_REF][START_REF] Park | p53-independent elevation of p21 expression by PMA results from PKC-mediated mRNA stabilization[END_REF]. Importantly, PKC has been convincingly implicated in COX-2 mRNA stabilization in several recent studies [START_REF] Doller | Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2[END_REF][START_REF] Doller | Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA[END_REF][START_REF] Subbaramaiah | Regulation of cyclooxgenase-2 mRNA stability by taxanes: evidence for involvement of p38, MAPKAPK-2, and HuR[END_REF][START_REF] Doller | Angiotensin II induces renal plasminogen activator inhibitor-1 and cyclooxygenase-2 expression posttranscriptionally via activation of the mRNA-stabilizing factor human-antigen R[END_REF][START_REF] Doller | Tandem phosphorylation of serines 221 and 318 by protein kinase Cdelta coordinates mRNA binding and nucleocytoplasmic shuttling of HuR[END_REF] and, indirectly, in IL-6 mRNA stabilization in one older study [START_REF] Akashi | Role of lymphotoxin in expression of interleukin 6 in human fibroblasts. Stimulation and regulation[END_REF]. In astrocytes, PKC was previously shown to be important for IL-6 induction after IL-1β [START_REF] Norris | Signal transduction pathways mediating astrocyte IL-6 induction by IL-1 beta and tumor necrosis factor-alpha[END_REF]. Moreover, IL-1β has been shown to activate several PKC isoforms, such as PKC-δ [START_REF] Limatola | Interleukin 1-beta-induced protein kinase C-zeta activation is mimicked by exogenous phospholipase D[END_REF][START_REF] Ganz | PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells[END_REF], PKC-γ [START_REF] Lin | Involvement of protein kinase Cgamma in IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells[END_REF], PKC-δ [START_REF] Carpenter | Protein kinase Cdelta activation by interleukin-1beta stabilizes inducible nitric-oxide synthase mRNA in pancreatic beta-cells[END_REF] and PKC-β [START_REF] Kracht | Interleukin-1-induced signaling in T-cells. Evidence for the involvement of phosphatases PP1 and PP2A in regulating protein kinase C-mediated protein phosphorylation and interleukin-2 synthesis[END_REF], in some of these studies in the context of mRNA stabilization.

To the best of our knowledge we are the first to show that the mRNA-stabilizing effect of IL-1β on IL-6 mRNA can also be p38-independent and might instead be mediated via PKC.

However, the conclusion that the mRNA-stabilizing effect of IL-1β is late, and thus probably indirect, substantially complicated our attempts to identify the involved PKC isoform. Since PKC activation can be a very fast process, sometimes disappearing already after a few minutes [START_REF] Ohmori | Three distinct mechanisms for translocation and activation of the delta subspecies of protein kinase C[END_REF], and since the time frame in which PKC could be activated in our experimental system ranged from approximately 4 to 6 hours, it unfortunately became virtually impossible to identify the responsible PKC isoform. Moreover, the existence of 10 different PKC isoforms complicates a traditional siRNA-based approach. Although the fact that PMA mimicked the effect of IL-1β indicates that the involved isoform belongs to the novel or classical subfamily, 8 isoforms are still left. The pharmacological PKC inhibitors that we used are not very suitable for distinguishing between the different PKC isoforms, since they both interact at the homologous ATP-binding site, and therefore they do not allow clear distinction between the different isoforms [START_REF] Way | Identification of PKC-isoform-specific biological actions using pharmacological approaches[END_REF]. Lastly, although the involvement of PKC in mRNA stabilization is well established [START_REF] Doller | Signalling pathways regulating nucleo-cytoplasmic shuttling of the mRNA-binding protein HuR[END_REF], and the pharmacological inhibitors we used have been intensively employed previously to investigate PKC involvement in a variety of processes, we cannot exclude that aspecific inhibition of another kinase might occur, causing inhibition of IL-1β-mediated IL-6 mRNA stabilization. For example, both Rsk-2 and p70 S6 kinases can be inhibited by both GF103209X and Ro31-8220 [START_REF] Alessi | The protein kinase C inhibitors Ro 318220 and GF 109203X are equally potent inhibitors of MAPKAP kinase-1beta (Rsk-2) and p70 S6 kinase[END_REF]. The involvement of these kinases in mRNA stabilization processes has however, to our knowledge, not yet been documented. A well characterized downstream target of PKC in mRNA stabilization processes is the AREbinding protein HuR [START_REF] Doller | Signalling pathways regulating nucleo-cytoplasmic shuttling of the mRNA-binding protein HuR[END_REF]. Moreover, the importance of the PKC-HuR axis in COX-2 mRNA stabilization is well established [START_REF] Doller | Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2[END_REF][START_REF] Doller | Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA[END_REF]. Nevertheless, the mRNA-stabilizing protein HuR, which has formerly been shown to bind the 3"UTR of IL-6 [START_REF] Nabors | HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine-and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs[END_REF], did not mediate IL-1βinduced mRNA stabilization of IL-6 in 1321N1 cells, although a constitutive stabilization of IL-6 mRNA did occur.

Whereas the actinomycin D experiments pointed to a very potent mRNA-stabilizing effect, IL-1β had only a modest, although highly reproducible and significant effect in the IL-6

3"UTR assay. This could mean that other elements in the IL-6 mRNA, such as introns or the 5"UTR, are involved in IL-6 mRNA stabilization after IL-1β treatment in astrocytes. On the other hand, in the IL-6 3"UTR assay, luciferase mRNA levels were assessed indirectly, via measurement of enzymatic activity of the translated luciferase protein. It could be that the mRNA-stabilizing effect in this assay is blurred by additional (post)translational mechanisms targeting the luciferase protein and we also cannot rule out that IL-1β affects the half-life of the luciferase protein.

Interestingly, in a recent paper, treatment of 1321N1 cells with β-adrenergic agonists inhibited their proliferation [START_REF] Toll | {beta}2-Adrenergic receptor agonists inhibit the proliferation of 1321N1 astrocytoma cells[END_REF]. In view of the known proliferative effects of IL-6 on brain glioma [START_REF] Saidi | Combined targeting of interleukin-6 and vascular endothelial growth factor potently inhibits glioma growth and invasiveness[END_REF][START_REF] Goswami | Interleukin-6-mediated autocrine growth promotion in human glioblastoma multiforme cell line U87MG[END_REF], this seems contradictory to the results we present here. This contradiction can however be The exact molecular cascade leading to IL-6 mRNA stabilization in our model system remains unclear. Nevertheless, we can hypothesize on several possible mechanisms, although we did not explore all of them. First of all, it was recently demonstrated that APOBEC-1 complementation factor (ACF) controls IL-6 mRNA stability in liver [START_REF] Blanc | Apobec-1 complementation factor modulates liver regeneration by post-transcriptional regulation of interleukin-6 mRNA stability[END_REF]. ACF is present in brain tissue [START_REF] Mehta | Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA[END_REF], so could be a potentially interesting target. Another recently identified novel mechanism involving IL-6 mRNA stabilization is the TLR-inducible RNase Zc3h12a [START_REF] Matsushita | Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay[END_REF].

Zc3h12a was shown to destabilize IL-6 mRNA after TLR-triggering, thus providing an essential mechanism for controlling immune reactions in vivo. Interestingly, Zc3h12a is also expressed in brain and was recently shown to be involved in glial differentiation of NT2 cells [START_REF] Vrotsos | MCP-1 involvement in glial differentiation of neuroprogenitor cells through APP signaling[END_REF]. Thirdly, alternative polyadenylation of IL-6 mRNA, i.e. the use of an upstream polyadenylation signal, resulting in a shorter mRNA, could possibly contribute to changes in mRNA stability. This mechanism has been convincingly described for COX-2 [START_REF] Hall-Pogar | Alternative polyadenylation of cyclooxygenase-2[END_REF] and HuR [START_REF] Al-Ahmadi | Alternative polyadenylation variants of the RNA binding protein, HuR: abundance, role of AU-rich elements and auto-Regulation[END_REF]. The human IL-6 gene contains an alternative polyadenylation signal less than 100 bp upstream from the canonical polyadenylation site [START_REF] Tanabe | Genomic structure of the murine IL-6 gene. High degree conservation of potential regulatory sequences between mouse and human[END_REF], and alternative polyadenylation would lead to the exclusion of several AU-rich regions in the alternative mRNA species. The possibility that alternative polyadenylation of the human IL-6 cDNA influences mRNA metabolism is intriguing, but remains uninvestigated up to now. Finally, AUF-1, another RNA binding protein, has been shown to bind IL-6 mRNA and to influence its stability [START_REF] Paschoud | Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element, and the RNA-binding protein AUF1[END_REF].

We have investigated if AUF-1 is involved in IL-1β-induced IL-6 mRNA stabilization by overexpressing the different AUF-1 isoforms (p37, p40, p42 and p45) in the IL-6 3"UTR A c c e p t e d M a n u s c r i p t reporter gene assay, but our preliminary data did not suggest a role for AUF-1 in this system (data not shown).

In conclusion, IL-1β stabilizes IL-6 and COX-2 mRNA via a novel, not yet completely characterized mechanism. This mRNA stabilization translates into a very potent synergistic IL-6 protein expression. Based on pharmacological inhibitor data, the stabilization process seems to depend on PKC, but not on p38, contrary to the earlier described modes of IL-6 mRNA stabilization [START_REF] Miyazawa | Regulation of interleukin-1beta-induced interleukin-6 gene expression in human fibroblast-like synoviocytes by p38 mitogen-activated protein kinase[END_REF][START_REF] Zhao | p38alpha stabilizes interleukin-6 mRNA via multiple AU-rich elements[END_REF][START_REF] Patil | p38 MAPK regulates IL-1beta induced IL-6 expression through mRNA stability in osteoblasts[END_REF][START_REF] Winzen | The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism[END_REF]. Moreover, the prototypical mRNA-stabilizing protein HuR is not involved. Finally, the effect is late and probably indirect, although it seems to be independent of protein secretion.

Further elucidation of the molecular mechanism underlying this novel mode of IL-1β induced mRNA stabilization is required, but in any case the very potent enhancement of IL-6 levels by combining two classical pro-inflammatory stimuli, TNF-α and IL-1β with a β-adrenergic agonist, isoproterenol, is bound to have profound physiological effects in vivo, specifically in the context of gliomas and neurodegenerative diseases. Moreover, the finding that PKC inhibitors are powerful tools to reduce astrocytic IL-6 expression might have interesting therapeutic implications.

Fig. 5

Effect of kinetic preinduction on IL-6 mRNA degradation rate. a-b) 1321N1 cells were preinduced with isoproterenol (10 μM) and TNF-α (2000 IU/ml) (iso+TNF) or isoproterenol (10 μM), TNF-α (2000 IU/ml) and IL-1β (2 ng/ml) (iso+TNF+IL) for 1, 3 or 6 hours. Subsequently iso, TNF-α and IL-1β were removed and actinomycin D (5 μg/ml) was added to block new transcription. mRNA was isolated at different time points after addition of actinomycin D (0, 60, 120 and 180 min) and subjected to RT-qPCR. Data are means of 3-4 independent experiments. ns P > 0.05 between iso+TNF versus iso+TNF+IL at that time point; a P < 0.05 versus iso+TNF at that time point. c) Effect of blocking protein secretion using brefeldin A (BFA) on IL-6 mRNA degradation rate. 1321N1 cells were preinduced for 30 min with or without BFA (5 μg/ml) and subsequently induced with isoproterenol (10 μM) and TNF-α (2000 IU/ml) (iso+TNF) or isoproterenol (10 μM), TNF-α (2000 IU/ml) and IL-1β (2 ng/ml) (iso+TNF+IL) for 6 hours. Iso, TNF-α, IL-1β and BFA were removed and actinomycin D (5 μg/ml) was added to block new transcription. mRNA was isolated at different time points after addition of actinomycin D (0, 60, 120 and 180 min) and subjected to RT-qPCR. Data shown are means of 2 independent experiments. ns P > 0.05 between iso+TNF+IL versus iso+TNF+IL+BFA for the corresponding time points; a P < 0.05 versus iso+TNF+IL at that time point. d) Effectiveness of brefeldin A. Cells were treated with BFA (5 μg/ml) for 6,5 hours and fixed with formaldehyde. Immunofluorescence staining was performed using an antibody for a Golgi marker (anti-GM130) to visualize the Golgi compartment.

A c c e p t e d M a n u s c r i p t 9

 9 Mowiol (Calbiochem, Gibbstown, MJ). Samples were analyzed using an Axiovert 200M Zeiss Microscope (Thornwood, NY).

  A c c e p t e d M a n u s c r i p t induction, we observed a very clear stabilizing effect of IL-1β. Double induction with isoproterenol and TNF-α yielded IL-6 mRNA with a half-life of approximately 60 minutes.

14 3. 4

 144 A c c e p t e d M a n u s c r i p t The effect of IL-1β seems to be mediated by PKC and not by p38.

  A c c e p t e d M a n u s c r i p t 15

A

  c c e p t e d M a n u s c r i p t 22 explained by, on the one hand, the differences in timing of the treatment with β-adrenergic receptor agonists (20 hours by Toll et al. and 6 hours in this paper); and on the other hand, the fact that IL-1β or TNF-α might possibly overrule the effects isoproterenol has on 1321N1 proliferation.

Synergistic

  Figure captions

Fig. 2

 2 Fig.2

A

  c c e p t e d M a n u s c r i p t a-e) After induction of 1321N1 cells for 30 min with vehicle (veh), isoproterenol (iso, 10 M), TNF-α (TNF, 2000 IU/ml) and IL-1β (IL, 2 ng/ml) individually or combined, cells were lysed in SDS sample buffer and subjected to Western analysis. Activation of p38 (a), JNK (b), ERK (c), CREB (d) or p65 (e) pathways was assessed using phospho-specific antibodies and tubulin as a loading control. Three independent experiments were used for quantification of Western blots. ns P > 0.05 versus iso+TNF. f) A representative blot after treatment of 1321N1 cells for 30 min as described under a-e). g) Effect on nuclear translocation of p65. 1321N1 cells were induced with isoproterenol (10 M) , TNF-α (2000 IU/ml), IL-1β (2 ng/ml) or combinations thereof for 30 minutes. Subsequently nuclear extracts were subjected to Western blot analysis to determine p65 levels. Data shown are representative for 3 independent experiments. h) IL-6 promoter activation after treatment with isoproterenol, TNF-α and IL-1β. 1321N1 cells were transiently transfected with an IL-6 promoter construct (1168-IL-6-luc). Reporter gene activation was measured with a luciferase reporter gene assay after induction for 6 hours with isoproterenol (iso, 10 M), TNF-α (TNF, 2000 IU/ml) and IL-1β (IL, 2 ng/ml), individually or combined. Data shown are means of 5 independent experiments. a P < 0.05 versus all individual inductions (veh, iso, TNF, IL);. ns P > 0.05 for iso+TNF versus iso+TNF+IL. i) Comparison of the ratio of triple (iso+TNF+IL) versus double (iso+TNF) induction at mRNA and promoter level. Mean ratios were calculated on the basis of 3-4 independent experiments, performed as described under Fig. 1b (mRNA level) and Fig. 2h (promoter level). a P < 0.05 versus promoter; ns P > 0.05 in one sample t test with the theoretical mean of 1.

Fig. 3

 3 Fig.3

A c c e p t e d M a n u s c r i p t 37 d

 37 ) COX-2 mRNA expression is synergistically induced in 1321N1 cells by combinations of isoproterenol (iso, 10 μM), TNF-α (TNF, 2000 IU/ml) and IL-1β (IL, 2 ng/ml). Triple (iso+TNF+IL) versus double (iso+TNF) induction clearly induces a "super"synergy. Cells were induced for 6 h, after which RT-qPCR analysis was performed. Data shown are mean values of 3 independent experiments. a P < 0.05 versus veh; b P < 0.05 versus iso+TNF.

Fig. 4

 4 Fig. 4

A

  c c e p t e d M a n u s c r i p t b) Effectiveness of SB203580. 1321N1 cells were induced for 30 min using isoproterenol (10 μM), TNF-α (2000 IU/ml) and IL-1β (2 ng/ml) (iso+TNF+IL) with or without SB230580 (10 μM). Lysates were subjected to Western analysis. Activation of MSK was assessed using a phosphospecific antibody (Anti-P-MSK (Thr 581)). c) Effect of a p38 inhibitor (SB) and a PKC inhibitor (Ro) on the mRNA degradation rate of IL-6, determined using actinomycin D. 1321N1 cells were pretreated for 6h with isoproterenol (10 μM)+TNF-α (2000 IU/ml) (iso+TNF) or isoproterenol (10 μM)+TNF-α (2000 IU/ml)+IL-1β (2 ng/ml) (iso+TNF+IL) in combination with SB203580 (SB, 10 μM) or Ro31-8220 (Ro, 5 μM)). Subsequently, iso, TNF-α, IL-1β, SB and Ro were removed and actinomycin D (5 μg/ml) was added to block new transcription. mRNA was isolated at different time points after addition of actinomycin D (0, 60, 120 and 180 min) and subjected to RT-qPCR. Data shown are mean values calculated from 3 independent experiments. a P < 0.05 iso+TNF+IL+Ro versus iso+TNF+IL at that time point; ns P > 0.05 between iso+TNF+IL versus iso+TNF+IL+SB at the corresponding time points. d) PMA mimics IL-1β-induced stabilization of the IL-6 3"UTR reporter gene. 1321N1 cells were transiently transfected with an IL-6 3" UTR construct (luc-IL-6 3"UTR). Reporter gene expression was measured with a luciferase reporter gene assay after induction for 6 h with vehicle (veh) or PMA (1 μg/ml). Data shown are mean values calculated from at least 5 independent experiments. a P = 0.0021 in a two-tailed t test versus veh. e) PMA mimics IL-1β-induced "super"synergistic IL-6 mRNA expression. IL-6 mRNA expression is synergistically induced in 1321N1 cells by combinations of isoproterenol (iso, 10 μM), TNF-α (TNF, 2000 IU/ml), IL-1β (IL, 2 ng/ml) and PMA (1 μg/ml). Triple induction with iso+TNF+PMA mimics the triple induction with iso+TNF+IL-1β. Cells were induced for 6 h, and RT-qPCR analysis was performed. Data shown are mean values of 3 independent experiments. a P < 0.05 versus iso+TNF; ns P > 0.05 for iso+TNF+PMA versus iso+TNF+IL.

Fig. 6

 6 Fig. 6

Figure1

  Figure1Click here to download high resolution image

Figure 2

 2 Figure 2 Click here to download high resolution image

Acknowledgements

The authors would like to thank Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO) for financial support. A.S. and K.K. are predoctoral FWO fellows and S.G. is a postdoctoral FWO fellow. P.M. is supported by the Ghent University Research Fund (BOF 01D31406). P.R. is a postdoctoral BOF fellow (Bijzonder Onderzoeksfonds). The authors are very grateful to Dr. Doller (Goethe-universität, Frankfurt am Main) for the generous gift of the FLAG-HuR plasmid, to Prof. Gorospe (NIH, Baltimore) for the generous gift of the AUF-1 expression plasmids, to Prof. Saito (University of Kobe), Prof. Ferguson (University of Western Ontario), Prof. Larrson (Lund University) and Dr. Doller (Goethe Universität) for the very generous gifts of the PKC-GFP expression plasmids, to Prof. Kirkwood (University of Michigan) for the generous gift of the IL-6 3"UTR-luciferase vector and to Prof. Muller (University of Bonn) for the generous gift of the 1321N1 cells. The authors would also like to thank R. Dahl.


Verpoest

S Daggumati

W Van Paepegem

J Degrieck

J Xu

S V Lomov

I Verpoest

Local damage in a 5 -harness satin weave composite under static tension: part ii -meso -FE modelling

Keywords: Textile composites, Finite element analysis (FEA), Damage mechanics, Weft yarn damage, Multiscale modeling

Local damage in a 5 -harness satin weave composite under static tension: part ii -meso -FE modelling

Introduction

In order to estimate the homogenized elastic constants of textile composites using unit cell meso-FE simulations, a numerical procedure is firmly established [START_REF] Carvelli | A homogenization procedure for the numerical analysis of woven fabric composites[END_REF][START_REF] Terada | Simulation of the multi-scale convergence in computational homogenization approaches[END_REF]. However, the meso-FE procedure still needs to be improved for analysis of the local stress and damage behavior of the composite using the unit cell FE simulations [START_REF] Ivanov | Damage analysis in textile composites[END_REF][START_REF] Carvalho | Compressive failure of 2D woven composites[END_REF]. The majority of the published numerical work regarding the analysis of local structural behavior in the textile composite is based on the single unit cell FE simulation with 3D PBCs (Periodic Boundary Conditions). The underlying principle in the application of 3D PBCs is that the unit cell is chosen from the middle of the laminate [START_REF] Lomov | Meso-FE modelling of textile composites: Road map, data flow and algorithms[END_REF][START_REF] Whitcomb | Three-Dimensional Stress Analysis of Plain Weave Composites[END_REF][START_REF] Whitcomb | Analysis of New Composite Architectures[END_REF][START_REF] Bogdanovich | Multi-scale modeling, stress and failure analyses of 3-D woven composites[END_REF], which represents the entire stress/strain fields in the composite.

However, recent publications [START_REF] Ivanov | Stress distribution in outer and inner plies of textile laminates and novel boundary conditions for unit cell analysis[END_REF][START_REF] Owens | Effect of Finite Thickness and Free Edges on Stresses in Plain Weave Composites[END_REF][START_REF] Lomov | Nesting in textile laminates: geometrical modelling of the laminate[END_REF] on the meso-FE analysis of the unit cell have emphasized the effect of free surface and free edges on local stress behavior. Moreover, the influence of internal yarn shifting on the local stress behavior of the unit cell has been highlighted. In order to investigate the effect of local fabric geometries on the damage behaviour, Le Page et al. [START_REF] Page | Finite element simulation of woven fabric composites[END_REF] developed two dimensional plane strain FE models of woven composite with in-phase, out-of-phase and staggered nested unit cell models. The above mentioned work concluded that the strain energy release rate associated with the crack formation is influenced significantly by the crack location. In addition, the formation of a crack is associated with the local bending deformation, and the energy release rate increases with the degree of bending. According to Adams et al. [START_REF] Adams | Idealized textile composites for experimental/analytical correlation[END_REF], the modelling approaches assume idealized textile architecture and generally consider a single unit cell. Due to the randomness of textile architecture produced using conventional processing techniques, the experimental data obtained has shown limited use for verifying the accuracy of these numerical models.

In the above context, Part I of this paper described the experimental observations of the sequence of damage events in different layers of the 5-harness satin weave carbon-PPS laminate, which are influenced by the local fabric geometries. Weft yarn transverse damage is initiated in the inner layers of the laminate, followed by damage on the surface layers. The sequence of damage events in different layers implies that the damage initiation may have been influenced by the local constraints imposed by the surrounding plies. In Part II of this paper, the above experimental observation is verified using the unit cell as well as laminate level 3D meso-FE simulations. The work presented here is conducted in the view of obtaining extensive knowledge on the mechanics of the local structural behaviour of a satin weave composite.

Estimation of the homogenized elastic constants

Construction of the unit cell FE model

In order to begin with the homogenization of elastic constants and the simulation of the local structural behavior on the meso scale, the textile reinforcement under consideration should be accurately constructed and translated to the FE software. The micro-CT technique is employed to investigate the variability of the internal yarn dimensions in a processed composite. To quantify the variation of the internal yarn dimensions in multiple composite samples at the same time, three laminates were taped together each with the dimensions of 1, the unit cell geometric model of the 5 -harness satin weave reinforcement is generated using the 'WiseTex' software [START_REF] Lomov | Textile composites: modelling strategies[END_REF][START_REF] Verpoest | Virtual textile composites software WiseTex: Integration with micro-mechanical, permeability and structural analysis[END_REF]. Later, the 'WiseTex' generated textile reinforcement is transferred into FE mesh and filled with the matrix using the 'MeshTex' software [START_REF] Lomov | Meso-FE modelling of textile composites: Road map, data flow and algorithms[END_REF]. Finally, the 'MeshTex' output in the form of nodal and elemental information is transferred to the ABAQUS software.

Homogenization of the elastic constants

In order to start the unit cell FE analysis, the micro-mechanical (UD) material properties of the carbon-PPS representing the homogenized material properties of the impregnated yarn are derived from the individual elastic properties of the carbon fibre and PPS matrix (Table 2) using the analytical Chamis [START_REF] Chamis | Mechanics of Composite Materials Past, Present, and Future[END_REF] homogenization formulas. The intra-yarn volume fraction ( f φ ) used for the calculation of the homogenized material properties (Table 3) is 70% (constant through out the yarn sections), which is obtained from the 'WiseTex' software. The analytical homogenized elastic constants of the impregnated yarn are compared with the CCA [START_REF] Hofstee | Elastic stiffness analysis of a thermo-formed plainweave fabric composite Part II: analytical models[END_REF] and show good correlation. The calculated homogenized micro-mechanical material properties are assigned to the yarn cross sections in their local co-ordinate system to account for the yarn crimp in the FE model.

Moreover, perfect bonding is assumed at the interface between the yarns and matrix of the unit cell: i.e. the yarn and the matrix share common node at the interface, which has been justified by Gerlach et al. [START_REF] Gerlach | The interface between matrix pockets and fibre bundles under impact loading[END_REF].

The effective elastic constants of the textile composite on the scale of a single unit cell are calculated using the numerical meso-mechanical homogenization procedure. Initially, the elastic constants of the composite on the meso scale are estimated by applying the Dirichlet boundary conditions to the unit cell. The procedure involved in the estimation of elastic constants using the Dirichlet boundary conditions is explained in [START_REF] He | Elastic constants estimation of stitched NCF CFRP laminates based on a finite element unit-cell model[END_REF][START_REF] Sun | Prediction of composite properties from a representative volume element[END_REF]. In order to calculate the homogenized elastic constants of the unit cell, FE analysis is started by solving 6 independent unit load cases (3 normal From this, nine homogenized elastic constants of the unit cell are computed (Table 4).

The other procedure used for the evaluation of the homogenized elastic constants of the unit cell is periodic boundary conditions (PBCs) [START_REF] Lomov | Meso-FE modelling of textile composites: Road map, data flow and algorithms[END_REF][START_REF] Pellegrino | Numerical homogenization of periodic composite materials with non-linear material components[END_REF][START_REF] Chapman | Effect of assumed Tow architecture on the predicted moduli and stress in woven composites NASA Contractor Report[END_REF][START_REF] Wang | Multi-scale Analyses of 3D Woven Composite Based On Periodicity Boundary Conditions[END_REF] along with the volume averaging technique. By applying the 3D PBC, the above mentioned six independent unit load cases are solved. Later, by the application of the volume averaging technique [START_REF] Carvelli | A homogenization procedure for the numerical analysis of woven fabric composites[END_REF][START_REF] Lomov | Meso-FE modelling of textile composites: Road map, data flow and algorithms[END_REF][START_REF] Carvelli | A micromechanical model for the analysis of unidirectional elastoplastic composites subjected to 3d stresses[END_REF], the homogenized elastic constants of the unit cell are estimated (Table 4). Finally, the elastic constants computed using different boundary conditions are compared to the analytical (method of inclusions [START_REF] Lomov | Homogenisation of a sheared unit cell of textile composite FEA and approximate inclusion model[END_REF]) and with the experimental elastic constants (Table 4).

The difference in the estimated values of the homogenized elastic constants between the periodic and Dirichlet boundary conditions has been explained by Terada et al. [START_REF] Terada | Simulation of the multi-scale convergence in computational homogenization approaches[END_REF]. In correlation with the observations of the current work, the homogenized elastic properties estimated using Dirichlet boundary conditions have higher predicted values than those of the periodic boundary conditions. From the elastic constants computed with different procedures, it is evident that the periodic boundary conditions along with the volume averaging technique predict better results compared to the experimental results.

Unit cell FE analysis

To start with the meso-FE damage analysis, along with the micro-mechanical elastic properties, the strength properties of the carbon-PPS UD (Table 5) are derived from the individual strength properties of the carbon fibre and PPS matrix (Table 2) using the Chamis analytical strength homogenization formulas [START_REF] Chamis | Mechanics of Composite Materials Past, Present, and Future[END_REF]. Once the transverse isotropic strength properties to the yarns and the isotropic material properties of the matrix are assigned, for the damage analysis of the unit cell the following approach is employed.

Initially, the occurrence of damage in the unit cell element is detected using the Hoffmann failure criteria [START_REF] Tolson | Finite element analysis of progressive failure in laminated composite plates[END_REF]. However, this criterion cannot indicate the type of damage mode in the yarns, where the architecture of the fibre arrangement plays an important role. The modes of the damage are classified into four different types (Table 6) [START_REF] Zako | On the damage behavior of fiber reinforced composite materials[END_REF]. The mode L represents the fibre breaking, T and Z modes represent the transverse and shear cracking. As shown in Table 6, by calculating the corresponding stress-to-strength ratios for the different modes, we consider that the damage mode that is taking place is the one whose stress-to-strength ratio has the maximum value. The above mentioned failure criteria and the damage model is implemented into the commercial FE software ABAQUS using UMAT Fortran routines, which enable us to simulate the damage in the unit cell.

The results obtained from the FE modeling are compared with the experimental observations as follows:

1. Validation of the FE damage initiation strain with the damage initiation strain obtained from the experimental acoustic emission technique (Part I).

2. Finding the FE damage initiation location in the unit cell, which could be verified with the microscopically observed damage initiation location on the quasi-static tensile tested composite specimen (Part I).

Finally, the local stress profiles are plotted for various unit cell models to analyze the effect of unit cell stacking and the applied boundary conditions.

CASE I: (FE simulation of the damage in the laminate inner layers)

In order to simulate the stress and damage behavior inside the laminate, FE analysis is started with a single unit cell by applying 3D PBCs [START_REF] Lomov | Meso-FE modelling of textile composites: Road map, data flow and algorithms[END_REF][START_REF] Chapman | Effect of assumed Tow architecture on the predicted moduli and stress in woven composites NASA Contractor Report[END_REF], which corresponds to the unit cell located in the middle of the laminate. During the loading process, at <0.2%> of the applied average tensile strain in the warp direction (Fig. 1 X-direction), the damage initiation is detected at the edges of the weft yarn at the yarn crimp location by Hoffmann criteria (Figure 1c). Once the Hoffmann criteria detect the damage initiation in the element, for the direction of the damage, individual stress to strength ratios are calculated using the formulas shown in the Table 6. And the values are listed below for the damaged elements.

Mode L (

F F c L t L l σ 2 ) = Negligible Mode T ( F F c T t T t σ 2 ) = 0.23 Mode Z ( F F c Z t Z Z σ 2 ) = 0.08 Mode TZ 2 ) ( F S TZ TZ τ = 0.16
Based on the individual stress to strength ratios shown above, the local transverse damage (meso level) in a satin weave weft yarn is a combination of the micro level transverse damage mode in combination with the out-of-plane shear damage mode. The predicted FE micro level damage modes are in correlation with the theory of Cox [START_REF] Cox | Handbook of Analytical Methods for textile composite[END_REF] on the satin weave composite architecture. Cox reported that the asymmetric satin weave fabric causes the coupling of bending and stretching, and there is also coupling between stretching and in-plane shear under the pure tensile load. In the present case, it is observed that the load coupling between stretching and in-plane shear is negligible compared to the load coupling between bending and stretching.

From the transverse stress contours (Figure 1a), the maximum stress of 49 MPa is observed at the damaged elements of the weft yarn. Moreover, the transverse stress profile (Figure 1b) is plotted on the surface elements of the weft and warp yarns between the yarn crimp locations as marked in Figure 1a. From the Figure 1b, the variation in local transverse stress is divided into three major parts as explained below:

• Starting from the centre of the weft yarn at the yarn crimp location, the transverse stress value increases from 30 MPa to the maximum value of 49 MPa at the edge of the weft yarn.

• The second phase of the stress profile starts at the edge of the weft yarn, and decreases to the minimum value (12 MPa) in the geometrical transition location between the crimp region to the flat position of the load carrying warp yarn.

• The third phase in the stress profile is related to the constant transverse stress of 10 MPa at the flat position of the load carrying warp yarn. In addition, the stress profile varies periodically along the unit cell geometry.

CASE II: (FE simulation of the damage on the laminate surface layer)

The current section deals with the numerical simulation of the local stress and damage behavior of the unit cell on the traction free surface. In order to understand the variation in local damage behavior in the finite surface laminate compared to the infinite laminate, the single unit cell FE analysis with 3D PBCs is changed as explained below.

Initially, the surface stress analysis is performed using the single unit cell with in-plane PBCs. The top and bottom surfaces of the unit cell (Figure 2a) are free to deform in the out-of-plane direction. Under the above specified constraints, at <0.25%> of the applied average strain in the warp yarn direction, weft yarn damage is detected at the yarn crimp location (Figure 2b). From the local stress analysis, it is observed that the maximum tensile stress (80 MPa) occurs at the centre of the weft yarn (Figure 2a). Due to the absence of textile reinforcement or supporting boundary conditions in the out-of-plane direction, the flat part of the warp yarn adjacent to the yarn crimp location is slightly compressed in the transverse direction (Figure 2c).

Comparison of the experimental (<0.6%>) and numerical (<0.25%>) surface damage initiation strains underline the necessity for improvement in the unit cell model used for the surface stress analysis. To capture the effect of underlying layers on the surface stress profile, it is necessary to create a unit cell stack for the FE analysis. However, producing the unit cell stack with the same mesh size as single unit cell is computationally expensive. In this regard, comparison of the numerical local strain profiles with different mesh densities to the experimentally measured strains by Lomov et al. [START_REF] Lomov | Full-field strain measurements for validation of meso-FE analysis of textile composites[END_REF] lead to the conclusion that, the effect of mesh size is minimal on the local strain variation. In the current work, to study the effect of mesh size on the local stress behavior, Figure 2c compares the local transverse stress profiles on the traction free surface with different unit cell mesh densities. The unit cell model with a coarse mesh has 8400 elements, and the fine mesh has 53200 elements. From the local stress curves (Figure 2c), it is evident that the maximum and minimum stress locations are the same. Further, there is a difference of approximately 10MPa stress between the fine and coarse mesh at the yarn crimp location.

In the process of improving the unit cell FE analysis, eight unit cells are stacked such that all the yarn crimp locations are in the same phase (Figure 3a). In-plane PBCs are applied to the edges of the unit cell, while the top and bottom surfaces are free to deform. With the aforementioned loading conditions, at <0.44%> of the global tensile strain in the warp yarn direction, transverse damage is detected at the weft yarn centre in the surface layer of the unit cell (Figure 3b). In addition, the transverse stress of 75 MPa is observed in the damaged elements (Figure 3a).

Results and discussion

In this section, comparison between the experimental (Part I) and numerical damage analysis is presented. This section is later extended to the analysis of unit cell local stress behavior at different locations of the laminate. The experimental AE (Acoustic Emission) technique predicts the weft yarn damage in the laminate inner layers around <0.1-0.2%> of the global tensile strain in the warp yarn direction. Simulation of the single unit cell with 3D PBCs predicts the weft yarn damage around <0.2%> of the global tensile strain, which proves to be a good correlation with the experimental results considering the fact that the computational model did not consider the thermal stress in the composite. In a sense, the damage initiation strain obtained from the FE simulations is a deterministic maximum value, which is within the limits of the experimental prediction. Moreover, the damage initiation location obtained from the FE simulation at the edge of the weft yarn (Case-I) shows a good correlation with the observed microscopic damage locations.

On the traction free surface, the single unit cell with in-plane PBCs and the 8 unit cell inphase stack predict the weft yarn damage at around <0.25%> and <0.44%> of the global tensile strain respectively. Comparison of the numerical damage initiation strain with the microscopically observed damage initiation strain on the surface (around <0.6%>) proves that the FE simulations predict the early damage initiation strain. In correlation with the experimental damage location, the numerical simulations predict the damage at the centre of the weft yarn. Based on the comparison of the experimental and numerical results, it is evident that without the presence of internal yarn shifting (nesting), by in-phase stacking of the yarn crimp locations, the maximum out-of-plane deformation in the surface layers is reached earlier than in the experimental counterpart (with the internal yarn nesting) and hence causes the early damage initiation [START_REF] Carvalho | Compressive failure of 2D woven composites[END_REF][START_REF] Ivanov | Strain mapping analysis of textile composites[END_REF].

In addition to the local damage analysis, the unit cell local stress analysis provides insight into the active stress components and their variations depending on the lamina position in the laminate. The major local stress components observed in the weft yarn at the yarn crimp location are the in-plane transverse stress ( 22σ ), the out-of-plane normal stress ( 33 σ ) and the out-of-plane shear stress ( 23σ ) components for the applied global tensile strain in the warp yarn direction. The local weft yarn stress values at the yarn crimp location for the single unit cell as well as the laminate are listed in Table 7. Comparison of the stress components inside the laminate (Case I) with the surface weft yarn stresses (CASE II) shows that the restriction of the yarn crimp inside the laminate causes a higher out-of-plane shear ( 23σ ) as well as out-of-plane normal stress ( 33 σ ) compared to the surface layers. The stress pattern observed above is in correlation with the research output of Lee et al. [START_REF] Lee | Digital phase-shifting grating shearography for experimental analysis of fabric composites under tension[END_REF] on the local strain behavior of a plain weave composite. The authors reported that the tension/in-plane shear and tension/bending coupling effects at the yarn crimp inside the laminate are restricted by the surrounding plies. In contrast, the unrestricted yarn crimp on the surface layers of the laminate caused weft yarn damage due to the maximum transverse and shear stress.

Apart from the local stress and damage analysis, studying the deformation behavior of the unit cell under the external load provides knowledge on the effect of stacking and the applied boundary conditions for the unit cell FE analysis. The literature [START_REF] Zako | On the damage behavior of fiber reinforced composite materials[END_REF][START_REF] Summerscales | Observations on the fibre distribution and fibre strain in a woven fabric reinforcement[END_REF][START_REF] John | Longitudinal and transverse damage taxonomy in woven composite components[END_REF] has shown that the tensile load applied to the unit cell causes the straightening of the yarn in the loading direction (warp). This straightening effect imposes the out-of-plane deformation on the perpendicular weft yarn. When the weft yarn deformation reaches its maximum value, damage will occur on the surface of the weft yarn. Based on the above statement, the out-of-plane deformation is plotted on the surface of the unit cell in the positive z-direction between two yarn crimp locations (dotted line in Figure 1a) at the moment of damage initiation. Considering the absolute difference from maximum to minimum deformation, as expected, the out-of-plane deformation is restricted with 3D PBCs (Figure 4a) causing an almost flat deformation starting from one yarn crimp to the other (peak points in the curves represent the yarn crossover position). In contrast, the single unit cell with in-plane PBC predicts a large difference in the out-of-plane deformation (Figure 4b) between the yarn crimp and the flat yarn position. Moreover, the absolute difference in the out-of-plane deformation value for the 8 unit cell stack (Figure 4c) at <0.44%> of the global strain is approximately twice the value of the infinite laminate at <0.2%>. The aforementioned statement shows that, by increasing the number of unit cells in the stack, the out-of-plane deformation at the yarn crimp of the finite surface unit cell stack approaches the infinite laminate deformation [START_REF] Ivanov | Stress distribution in outer and inner plies of textile laminates and novel boundary conditions for unit cell analysis[END_REF].

Conclusions

Part I of this paper introduced an experimental procedure for analyzing the damage initiation and the history of the damage in a 5-harness satin weave composite. The experimental procedure provided the qualitative as well as quantitative knowledge about the damage. In order to understand the stress behavior and the mechanics of damage, the current paper deals with the satin weave unit cell computational model. Numerical simulations provided insight into the mesoscopic damage locations and the stress behavior. Comparison of the experimental and simulation results lead to the following conclusions:

• The predicted FE damage initiation strain and location for the inner layers of the laminate are within the limits of the experimental observation. On the other hand, the single unit cell with 2D PBC and in-phase stacking of an 8 unit cell laminate predict the early damage initiation strain of <0.25%> and <0.44%> on the surface layers, compared to the experimental damage initiation of <0.6%>. This early damage initiation strain in the FE simulation can be attributed to the absence of internal yarn shifting in the computational model.

• The local damage initiation trend obtained from the numerical simulations demonstrates that the infinite laminate predicts early damage initiation at the edges of the weft yarn. In contrast, the free surface unit cells predict damage in the later stage.

• In correlation with the experimental observations, the different weft yarn damage initiation strains at different locations of the laminate suggest that the transverse weft yarn damage in a textile composite is a sequential process.

• In conclusion, the effect of internal yarn shifting (nesting) on the local structural response cannot be neglected and can not be captured by the infinite laminate FE analysis.

The essence of the two parts of the current local damage analysis is summarized as follows. By observing the experimental microscopic damage phenomena on the scale of the laminate at the end of the loading process, formation of cracks in the weft yarn at different locations appear to be completely stochastic in nature. However, by segregating the entire damage mechanism at different length scales, analyzing the factors contributing to the damage on the relevant scale, following the history of the damage and using the numerical simulations, the failure mechanism in textile composites can be understood in a certain deterministic way.

  and this stack was used for the micro-CT analysis. The size of the composite samples used for the micro-CT is determined according to the optimum dimensions of the unit cell. The output micro-CT images were reconstructed and used for the measurement of the textile parameters. The textile information required for the construction of unit cell geometry such as the spacing, width and thickness of the yarns are measured at 20 different locations in both the warp and weft directions of the micro-CT images. The averaged values are shown in

[

  ABD stiffness matrix. By inverting the ] [ AD stiffness matrix, the compliance components of the unit cell were obtained.

Figure 1 .Figure 2 .Figure 4 .

 124 Figure 1. Single unit cell with infinite laminate boundary conditions : a) transverse stress distribution on the weft yarn; b) transverse stress profile between two yarn crimp; c) transverse damage on the weft yarn at the yarn crimp location <0.2%>.

Table

Table 1 . Textile parameters obtained from the micro-CT measurements.

 1

	WiseTex input data at fabric level
	Property	5 harness satin weave warp 5 harness satin weave weft
	Yarn	198 tex
	Number of measurements	20	20
	Yarn width (mm)	1.31 ± 0.01	1.32 ± 0.08
	Yarn thickness (mm)	0.162 ± 0.01	0.161 ± 0.06
	Yarn spacing (mm)	1.50 ± 0.04	1.49 ± 0.07
	WiseTex input data at fibre level
	TEX (g/km)	198
	Yarn filament count	3000
	Filament diameter (mm)	0.007
	Carbon fibre density (g/cm 3 )	1.75

Table 2 . T300 JB carbon fibre, PPS elastic and strength properties.

 2

	T300 JB carbon fibre		PPS (PolyPhenelyene Sulphide)
	Elastic properties [5, 34]
	Longitudinal modulus 11 E , GPa	231	Modulus of elasticity E , GPa	3.8
	Transverse modulus 22 E , GPa	28	Shear modulus G , GPa	1.38
	In-plane shear modulus 12 G , GPa	24	Poisson's ratio ν	0.37
	Transverse shear modulus 23 G , GPa	10.7
	In-plane Poisson's ratio 12 ν	0.26
	Transverse Poisson's ratio 23 ν	0.39
	Strength properties
	Tensile strength, MPa	4210	Tensile strength, MPa	90
	Compressive strength, MPa	2616 Compressive strength, MPa	148
	Shear strength, MPa	-NA-	Shear strength, MPa	63
	Note: T300 JB carbon material strength properties are taken from TORAYCA technical datasheet No:
	CFA-002.
	PPS material properties are taken from TECHTRON & RYTRON PPS data sheet.

Table 3 . Homogenized elastic properties of the carbon-PPS impregnated yarn.

 3

	φ	f	=	0	.	7	11 E , [GPa]	, [GPa] 22 E	12 G , [GPa]	23 G , [GPa]	ν	12	ν	23
	Chamis micro
	mechanical	162.60	13.70	6.50	5.07	0.29	0.35
	homogenization
	CCA[17]
	Concentric Cylindrical	162.20	15.0	6.20	-	0.28	0.40
	Assemblage

Table 4 . Computed homogenized elastic constants of the carbon-PPS satin weave unit cell.

 4

			PBC and Volume averaging	Dirichlet BC's and reactions Surface	Method of (Analytical) Inclusions	Experiment
	11 E , GPa	56.49	59.5	61.7	57 ± 1
	22 E , GPa	56.41	59.5	61.7	-NA-
	33 E , GPa	10.53	10.55	10.56	-NA-
	ν	12	0.08	0.057	0.053	0.05 ± 0.02
	ν	13	0.41	0.41	0.44	-NA-
	ν	23	0.41	0.41	0.44	-NA-
	12 G MPa	4280	4305	4297	4175[35]
	13 G , MPa	3048	3286	3375	-NA-
	23 G , MPa	3045	3286	3375	-NA-

Table 5 . Homogenized strength properties of the carbon-PPS impregnated yarn.

 5

		Carbon-PPS
	Mechanical property	impregnated yarn ( f φ =0.7)
	Tensile strength, MPa

Table 6 . The characteristics of anisotropic damage model for fiber bundle and isotropic damage model for matrix[27].Table 7 . Local stress distribution on the weft yarn cross sections at the damage initiation strain.

 67

		In-plane transverse stress, MPa (edge/ centre of the yarn)	Normal out-of-plane stress, MPa (edge/ centre of the yarn)	Out-of-plane shear stress, MPa (edge/ centre of the yarn)
	CASE I (Inside the laminate)	49/31	14/14	22/2
	CASE II-Single unit
	cell-2D pbc (on the	49/78	Negligible	16/7
	surface)
	CASE II-8 unit cell stack (on the surface)	62/75	Negligible	4/18
	CASE II-8 unit cell
	stack (inside the	55/60	8/12	15/15
	laminate)

ACKNOWLEDGEMENTS

The authors would like to acknowledge the FWO -Vlaanderen for the financial support provided through the project G.0233.06H. The authors also express their gratitude to 'Ten Cate' for supplying the composite plates.