Hui Wang

Lorenzo Vendrame

Christophe Fliedel

Si Chen

Florence Gayet

Muriel Franck D'agosto

Eric Lansalot

Rinaldo Manoury

Poli

Dr Franck D'agosto

Dr Muriel Lansalot

Eric Manoury

Rinaldo Poli
email: rinaldo.poli@lcc-toulouse.fr

Mr Ms H L Wang

Dr C Vendrame

Dr S Fliedel

Dr F Chen

Dr E Gayet

Prof R Manoury

Triphenylphosphine-functionalized core-cross-linked micelles and nanogels with a polycationic outer shell: synthesis and application in rhodium-catalyzed biphasic hydrogenations

Keywords: Polyelectrolyte, RAFT polymerization, Polymerization-induced self-assembly, Poly(1-methyl-4vinylpyridinium iodide), Catalytic nanoreactors

Introduction

Transformations requiring expensive molecular catalysts (metals and/or ligands) can only be implemented at the industrial level if the catalyst can be efficiently separated from the reaction products and recycled. An attractive way to achieve this is through the liquid/liquid biphasic implementation, involving catalyst confinement in a different liquid medium than the reagents and products and allowing catalyst separation and recovery by decantation. Water is a particularly attractive medium for catalyst confinement because it is inexpensive and non-toxic. [START_REF] Cornils | Aqueous Phase Organometallic Catalysis[END_REF] The best-known aqueous biphasic industrial process is probably the Ruhrchemie/Rhone-Poulenc (RRP) rhodium-catalyzed hydroformylation of light olefin (propene, butene). [START_REF] Kohlpaintner | [END_REF] In liquid/liquid biphasic protocols, the catalyzed transformation may take place in one of four distinct environments: (i) in the catalyst phase, if the substrates have non-zero solubility in the catalyst medium (for instance, as in the RRP process); (ii) in the substrate/product phase, if the catalyst can be transported to that phase by a temperature stimulus (thermomorphic catalysis) [START_REF] Behr | Thermomorphic solvent systems[END_REF] or by a phase transfer agent; (iii) at the interface, if neither component is sufficiently soluble in the other component phase; [4] and finally (iv) within the homogeneous environment of catalytic nanoreactors such as functionalized micelles. In aqueous biphasic protocols, very hydrophobic substrates do not have sufficient solubility in water, leading to severe mass transport limitations for the implementation of type (i) and hence require using one of the other methods, (ii), (iii) or (iv). The last protocol has recently attracted considerable attention, because kinetically stable micelles can be easily formed by assembling amphiphilic diblock copolymers in water. [5] However, recognized limitations of this approach are micellar swelling, which leads to slow-decanting emulsions, and equilibria with single chains, which leads to catalyst leaching.

We have recently developed new polymeric supports for aqueous biphasic catalysis, consisting of core-crosslinked micelles (CCM) [6] and nanogels (NG), [7] see Figure 1. These are unimolecular nanosized polymeric architectures and thus immune from extensive swelling and particle-chain equilibria. The catalyst is covalently anchored within the hydrophobic core and the particles remain confined in the water phase thanks to a hydrophilic outer shell. The CCM and NG particles differ by the core architecture. In the former, amphiphilic diblock macromolecules with hydrophobic and ligand-functionalized linear chains are crosslinked at the end of the hydrophobic blocks in the final synthetic step, whereas the latter are built by simultaneous chain extension, functionalization and crosslinking from a hydrophilic macromolecular chain transfer agent. The main differences between CCM and NG is that the first one features crosslinking only in the central part of the core and the ligands are placed on flexible arms outside the crosslinked area, whereas the latter has an almost entirely crosslinked core with the ligands incorporated inside it. The technique that efficiently allowed us to develop particle with controlled size and architecture is reversible addition-fragmentation chain transfer (RAFT) polymerization, [8] implemented under aqueous conditions and resulting in "polymerization-induced selfassembly" (PISA). [9] Other types of unimolecular polymer-based catalytic nanoreactors, which are crosslinked at the level of either the outer shell, the core, or an inner corona, have also been developed by other groups, [10] though they were not employed under aqueous biphasic conditions with particular attention to catalyst leaching and recycling. Our first generation polymers were constructed with a neutral hydrophilic shell based on randomly copolymerized methacrylic acid (MAA) and poly(ethylene oxide) methyl ether methacrylate (PEOMA). The hydrophobic core consisted of polystyrene (PS), where the desired ligand was introduced by copolymerization of a suitably functionalized styrene (e.g. 4diphenylphosphinostyrene, DPPS, to yield PS-anchored triphenylphosphine functions) and diethylene glycol dimethacrylate (DEGDMA) was used as crosslinker. [6][7]11] After coordination of a suitable precatalyst, these polymeric nanoreactors proved efficient in the aqueous biphasic rhodiumcatalyzed hydroformylation of a model water insoluble α-olefin (1-octene) [6a, c, 7, 11a] and in the rhodium-catalyzed hydrogenation of 1-octene and styrene, [11d] and their recyclability was demonstrated. However, these systems had two limitations, negating their possible implementation for large scale applications: (i) after removal from the reaction medium (stirring at 1200 rpm at 80°C for two hours) the phase separation was rather slow, requiring several hours to yield an optically transparent organic phase; (ii) the presence of a significant Rh concentration in the organic phase was revealed by ICP-MS. The Rh leaching was demonstrated to be associated to the presence of polymeric nanoreactors in the recovered organic phase (DLS measurements) and not to loss of Rh molecules from the nanoreactor core. In addition, the amount of leached Rh was shown to be stirring-and architecture-dependent (1.7-2.5 ppm for CCM [6c] or 0.6 ppm for NG [7] at 1200 rpm; sharp leaching increase for the CCM as the stirring rate was increased up to 1600 rpm). [6c, 7] Furthermore, the DLS of the recovered organic phase showed that the nanoreactors in the organic phase had undergone irreversible agglomeration (more extensive for greater stirring rates) and were kinetically stabilized in the organic phase. The agglomeration phenomenon was investigated in greater detail and proven to be associated to a reversible particle-particle interpenetration and subsequent irreversible interparticle arm-arm coupling via ligand exchange at the Rh center. [11c] All these phenomena were recognized to be promoted by the temperature-sensitive behavior of the neutral P(MAA-co-PEOMA) blocks. At higher temperatures, the outershell becomes less hydrophilic, favoring transfer to the organic phase, and more lipophilic, favoring particle-particle interpenetration.

In order to correct these problems, we have thus developed a second-generation nanoreactor scaffold, containing a hydrophilic outer shell that does not suffer from temperaturesensitive behavior. As a first choice, we have selected a polycationic shell that contains quaternized (methylated) 4vinylpyridine (4VP) units, -[CH2-CH(4-C5H4NMe + I -)]-(4VPMe + I -). In a recent contribution, we have described the optimized synthesis of both CCM and NG particles with a PS hydrophobic core (without anchored ligand functionalities) and DEGDMAbased crosslinking. [12] After surmounting a few synthetic obstacles, particles of controlled architecture, narrow size distribution and quantitative arm crosslinking could be obtained.

In the present contribution, we report the synthesis and characterization of triphenylphosphine-functionalized versions of these particles (TPP@CCM and TPP@NG; see Figure 1), their coordination chemistry with [RhCl(COD)]2, an investigation of the interparticle metal migration, and finally their application in the aqueous biphasic hydrogenation of styrene and 1-octene as model substrates, demonstrating their superior performance in all respects (activity, speed of decantation, leaching) relatively to the first-generation nanoreactors.

Results and Discussion

(a) Polymer syntheses

The optimized syntheses of the polymers (see Figure 2) started from the diblock macroRAFT agent R0-(4VPMe + I -)140-b-S50-SC(S)SPr, already reported in our recent contribution, [12] which was obtained in three steps: (i) homogeneous RAFT polymerization of 4VP in water/ethanol with 4-cyano-4thiothiopropylsulfanyl pentanoic acid (CTPPA, or R0-SC(S)SPr with R0 = -C(CH3)(CN)CH2CH2COOH) as RAFT agent and 4,4'azobis(4-cyanopentanoic acid) (ACPA) as initiator, to yield R0-4VP140-SC(S)SPr; (ii) chain extension with styrene, which leads to PISA, with formation of a latex of R0-4VP140-b-S50-SC(S)SPr; (iii) reaction with MeI/DMF, leading to the desired macroRAFT agent, which was isolated as a yellow powder containing residual DMF of solvation (R0-(4VPMe + I -)140-b-S50-SC(S)SPr•34DMF). All steps led to narrowly distributed molar masses attesting the control of the polymerization. [12] Core-crosslinked micelle For the CCM synthesis, this macroRAFT agent was first chain-extended with the appropriate mixture of styrene and diphenylphosphinostyrene (DPPS), yielding latexes of the corresponding R0-(4VPMe + I -)140-b-S50-b-(S1-x-co-DPPSx)300-SC(S)SPr amphiphilic linear chains. Three versions were prepared with x = 0.05 (diblock 5%, with an average of 15 ligands per chain), 0.1 (diblock 10%, 30 ligands per chain) and 0.2 (diblock 20%, 60 ligands per chain). These polymers were then crosslinked with a DEGDMA/S (10:90) mixture in the final step to afford R0-(4VPMe + I -)140-b-S50-b-(S1-x-co-DPPSx)300-b-(S0.9-co-DEGDMA0.1)140-SC(S)SPr (CCM 5%, CCM 10% and CCM 20%, respectively). The latex of the NG particles (NG) was obtained in a single step, copolymerizing simultaneously styrene, the ligand-functionalized DPPS monomer and the DEGDMA crosslinker. Only a version containing on average 30 DPPS monomers per chain, R0-(4VPMe + I -)140-b-S50-b-(S426-co-DPPS30co-DEGDMA15)-SC(S)SPr, like the CCM 10% product, was developed, although the total amount of styrene and crosslinker per chain is greater and therefore the overall ligand concentration in the hydrophobic core is intermediate between those of CCM 10% and CCM 5%. All the latexes obtained were low viscosity colloidal dispersions, in spite of the high polymer content (16-24% in weight), with a milky aspect. They were all stable in time, giving no evidence of destabilization or gradient development over several months. The particle sizes were characterized by DLS and TEM and the corresponding polymers were characterized by 1 H and 31 P NMR spectrometry. The particle size was too large to allow meaningful SEC or NMR-DOSY analyses.

The 1 H NMR spectra obtained after diluting an aliquot of the latex into different deuterated solvents are collected in Figures S1-5. Dilution with DMSO-d6 allowed monitoring the monomer consumption but the particle core resonances were not revealed, since DMSO is not a good solvent for PS. On the other hand, after core swelling with CDCl3a good solvent for PSand dilution into D2O, the core resonances became observable. Like for the previously published latexes of the related CCM and NG particles with the neutral P(MAA-co-PEOMA) shell, [6c, 7] resonances that could be unambiguously attributed to the DEGDMA crosslinker could not be clearly distinguished, probably because of the small amount of this monomer. It is also worth pointing out that the DEGDMA protons, being close to the polymer crosslinking points, have more restricted mobility and their resonances are therefore expected to be broader. The DPPS incorporation in the polymer core cannot be assessed from the 1 H NMR spectra because the corresponding resonances cannot be distinguished from those of the styrene units. However, the presence of core-linked DPPS units was clearly indicated by a relatively broad resonance in the 31 P NMR spectrum at ca. -6.5 ppm, slightly shifted from the resonance of the precursor DPPS in the same CDCl3 solvent (Figure S6). The same broadening and resonance shift was previously observed for the incorporation of DPPS in the equivalent polymers with the neutral outer shell. [6c, 7] The DLS and TEM analyses (Figure 3) showed that all obtained polymers have spherical morphology, with average diameter in the 130-150 nm range and narrow size distributions. The zeta potentials are positive and as expected essentially equivalent, with experimental error, for all polymers. In addition, DLS measurements of CCM 5%, CCM 10% and NG after freeze-drying and redispersion in a DMSO-toluene 80:20 (v/v) mixture revealed the absence of uncrosslinked arms (see Figure S7). Indeed, this solvent mixture is able to solvate both blocks and would reveal the presence of single chains at smaller diameters, as previously demonstrated for the analogous DPPSfree diblock. [12] The particle diameters revealed by the TEM images are a bit smaller than those obtained from the DLS data (see the frequency analysis in Figure 3, bottom part), because the TEM measures the objects after deposition and drying on the grid support, whereas the DLS data are obtained on the solvated particles and thus reflect the diameter expansion by the hydrophilic shell solvation.

An exploration by TEM analysis (details in the SI, Table S1) of the CCM morphology dependence on the hydrophilic and hydrophobic block molar masses showed that a reduction of the hydrophilic block molar mass (72 or 56 4VPMe + I -units), while maintaining a similar molar mass for the hydrophobic part, led to mixtures of cylindrical and spherical micelles for the linear amphiphilic intermediate. This switch in morphology is consistent with the behavior of block copolymers with higher molar mass hydrophobic block and self-assembling during a PISA process. [9d] It indirectly attests the control of the polymerization during chain extension, indicating that well-defined block copolymers were achieved. Interestingly, rearrangement to spherical CCM particles occurred when using greater amounts of S/DEGDMA in the crosslinking step. The corresponding strong plasticization of the core of the nano-objects probably helps the reorganization of the block copolymers during this step into spherical morphologies, rapidly locked by the crosslinking reaction. Shortening the hydrophobic chain molar mass (e.g. R0-(4VPMe + I -)56-b-S32-b-(S1-x-co-DPPSx)118-) ensured a spherical morphology for the intermediate micelles, although the final crosslinked objects had a less well-defined morphology. The most homogeneous spherical morphology for both the intermediate micelles and the final CCM particles was obtained when using the longer outer block (140-150 4VPMe + I -monomer units). Since the spherical morphology seems most suitable for applications as nanoreactors in biphasic catalysis, the optimum balance of hydrophilic and hydrophilic parts of the CCM nanoreactors was fixed as ca. 150 and 300 before crosslinking, and an overall equivalent ratio of hydrophilic/hydrophobic parts was maintained for the NG synthesis.

In terms of swelling capacity and mass transport, these polymers do not show any substantial difference relative to the equivalent ones with the neutral P(MAA-co-PEOMA) outer shell: the incorporation of swelling solvents (toluene or chloroform) was quite rapid (< 1 min) upon shaking the biphasic mixture, as visually assessed by the change of the relative phase volumes, and gave rise to an average diameter increase while maintaining narrow size dispersions (Figure S8). Therefore, changing the hydrophilic shell from neutral P(MAA-co-PEOMA) to polycationic P4VPMe + I -does not negatively affect the migration of neutral organic compounds from an external continuous phase to the nanoreactor core. (b) [RhCl(COD)]2 precatalyst coordination and migration studies Using the same protocol optimized in previous contributions, [11d, 13] complex [RhCl(COD)]2 was introduced into the nanoreactor cores after pre-swelling the polymer particles with toluene. Upon equilibrating the latex phase with a toluene solution of the metal complex under vigorous stirring at room temperature, the Rh complex was transferred into the nanoreactor cores quantitatively within 30 min, as optically assessed by the transfer of the complex orange color from the organic to the aqueous phase. Hence, the polycationic nature of the outer shell also makes no opposition to the metal complex migration. Spectroscopic evidence of metal complexation, leading to the core-anchored [RhCl(COD)(TPP@CCM)] functions, was possible only when the polymers were quantitatively charged with the metal complex (P:Rh = 1:1). The free TPP resonance at -6.5 ppm was fully replaced by a doublet at 32.2 ppm assigned to the Rh-coordinated TPP (e.g. see the spectra for the CCM 10% particles in Figure 4). Although relatively broad because of the increased correlation time in the polymeric environment, the Rh coupling is clearly discernible with JPRh ≈ 150 Hz, consistent with previous studies. [6a, 7, 11a-c, 13] These spectral parameters agree well with those reported for the molecular model (31.5 ppm, J = 152 Hz) [14] and for the same complex anchored to the equivalent neutral-shell polymer (29.3 ppm, J = 150 Hz). [13] The spectra for the Rh-charged particles at higher P:Rh ratios (2:1 or 4:1) gave no observable signal because of further broadening by the rapid degenerative phosphine exchange, as previously described for the equivalent neutral-shell polymers. [13] In our previous work with the equivalent neutral-shell nanoreactors, the 31 P signal disappearance at higher P:Rh ratios could be used to demonstrate a fast metal migration between (a) (b) d/ppm cores of different macromolecules. A different complex, [Rh(acac)(CO)2], was used in that case, yielding [Rh(acac)-(CO)(TPP)] upon introduction in the CCM core. When two TPP@CCM latexes, one pristine and one 100% loaded with the Rh complex, were mixed in a 1:1 ratio (thus with a global P:Rh ratio of 2:1), the 31 P signals disappeared immediately. This phenomenon was proven to result from phosphine exchange reactions, [11c] by analogy with the behaviour in homogeneous solution, [15] which requires reversible particle interpenetration and core-core contact. Indeed, the migration was stopped at high pH (13.6), because the Coulombic repulsion between the polyanionic shells generated by deprotonation of the methacrylic acid units stops the interpenetration process. The present polycationic shell polymers proved to be equally capable of stopping the metal migration, as demonstrated by the persistence of the simultaneously observable 31 P NMR resonances for the free and coordinated TPP ligands in the 1:1 mixture of pristine and 100% loaded TPP@CCM latexes (the Pricher CCM 20% sample was used for this experiment), even after stirring the mixture for over 1 week at room temperature, see Figure 5. This demonstrates the efficient confinement of the metal complex within the core of the nanoreactor in which it has initially been anchored. Obviously, intraparticle phosphine exchange processes continue to take place, as shown by the absence of the 31 P resonance for partially loaded latexes. It is interesting to compare the monitoring of Figure 5 with that of the corresponding experiment for the polymer with the neutral P(MAA-co-PEOMA) shell at high pH. In the latter case, even though the immediate exchange via core-core contact was stopped, a slow change (complete in ca. 10 h at room temperature) took place with formation of a single product, [Rh(OH)(CO)(TPP@CCM)2], indicating a slower metal migration accompanied by a chemical transformation. This phenomenon was shown to involve Rh extraction from the polymer-bound [Rh(acac)(CO)(TPP@CCM)] complex by OH -and migration through the continuous aqueous phase, presumably as anionic [Rh(acac)(OH)(CO)] -. In the present case, the insignificant change of the NMR spectrum over 1 week indicates the absence not only of core-core contact but also of any metal migration through the continuous phase. Namely, the Rh metal does not leach out of the nanoreactor core.

(c) Biphasic catalytic hydrogenations

All nanoreactors were used to catalyze the hydrogenation of styrene and 1-octene as representative aromatic and aliphatic unsaturated substrates. Two different protocols were used for styrene, with the substrate introduced either neat or diluted into 1-nonanol. For 1-octene, the neat substrate could not be used because it is not a good solvent for the polystyrene core and is therefore not able to swell it. The catalytic application required substrate dilution into a good solvent for polystyrene. The choice of 1-nonanol was guided by our previous optimization study of the same biphasic catalytic reaction with the neutral-shell polymer, [11d] where a first attempt with use of toluene (a better solvent for polystyrene) led to coagulation because of particleparticle coupling. This phenomenon was interpreted as resulting from the nanoreactor interpenetration, allowing the coordinatively unsaturated Rh center in catalytic intermediates to bind to TPP ligands of different particles. Using 1-nonanol (sparingly soluble in water, good solvent for polystyrene, and able to stabilize transient unsaturated forms of the catalytically active species by coordination) led to successful implementation of the reaction with no polymer coagulation and rapid phase decantation. Although the polycationic shell of the new nanoreactors should stop the particle interpenetration, the use of 1-nonanol was initially maintained in order to compare the performances of the cationic and neutral shell polymeric nanoreactors.

c.1. Hydrogenation of styrene in 1-nonanol.

A first exploratory investigation was carried out to assess the effect of various parameters on the catalytic efficiency: P:Rh ratio (1:1, 2:1 and 4:1, for the CCM 10% nanoreactor), P content in the CCM (5% vs. 10%) and nanoreactor architecture (CCM vs. NG). In all cases, decantation was very rapid, as previously observed for the equivalent hydrogenation with the neutral-shell nanoreactors. For each experiment, the only detected product was ethylbenzene, showing selective hydrogenation of the vinyl function as expected for a molecular Rh catalyst. The generation of Rh nanoparticles, which is known to occur from Rh I precursor in the absence of stabilizing π-acidic ligands, would also lead to significant ring hydrogenation, as shown in previous reports of the reduction of arenes [16] including styrene. [17] Using a 10% (v/v) styrene solution in 1-nonanol, a styrene/Rh ratio of 200, and an H2 pressure of 20 bar at 25°C, quantitative substrate conversion was achieved after ca. 5 h of stirring in all cases. A representative conversion vs. time plot for the CCM 10% system with a P:Rh ratio of 4:1 is shown in Figure 6 and the full data for all systems are collected in the SI (Table S2). Considering the need of single-point kinetic monitorings and the associated experimental errors, leading to a relatively large scatter of the data (see Figure S9), no clear trends can be derived from a comparison of the data obtained under different experimental conditions. Thus, all subsequent experiments made use of the CCM 10% scaffold with a P:Rh ratio of 4:1. From the initial slope in Figure 6, the TOF can be roughly estimated as 70 h -1 .

The performance of the CCM 10% biphasic system was also compared with the homogeneous system operated under identical conditions. The two systems differ only by the biphasic vs. monophasic nature, introducing the potential effect of mass transport on the reaction kinetics, and by the effective catalyst concentration.

Indeed, for the biphasic nanoreactor implementation, all the Rh active centers are concentrated within the nanoreactor cores, the total volume of which is much smaller than the total volume of the organic phase. From the polymer content in the latex, the nanoreactor swelling capacity and the amounts used in the catalytic experiments, the effectively catalyst concentration in the nanoreactors can be roughly estimated as 2.3 times greater than that of the homogeneous system. The transformation should therefore be faster for the biphasic nanoreactor implementation, because of the rate dependence on the catalyst concentration, in the absence of mass transport limitations. However, the chemical environment around the catalytic center is not exactly the same for the two systems: substrate/1-nonanol for the homogeneous system and substrate/polystyrene/1-nonanol for the biphasic one. A comparison of the results obtained for the three different P:Rh ratios (1:1, 2:1 and 4:1) shows an approximately equal reactivity, with complete conversions within ca. 2.5 hours (see Figure S10). Once again, each data point comes from a different experiment and thus errors may be large, preventing a more quantitative kinetics comparison. However, the fact that the biphasic system is not significantly faster than the homogeneous one suggests mass transport limitations. The present cationic-shell nanoreactors are more efficient than the equivalent neutral-shell CCM nanoreactors, since the latter gave quantitative conversions only after ca. 20 h under the same experimental conditions. [11d] We can conclude that the cationic shell allows faster transport of styrene toward the polymer core and/or of the ethylbenzene product back toward the continuous organic phase, relatively to the neutral P(MAA-co-PEOMA) shell. Another important parameter, which indeed motivated the development of this second-generation cationic-shell nanoreactors, is catalyst leaching. It was hoped that reduced lipophilicity of the polycationic P(4VPMe + I -) shell, relative to the neutral-shell first-generation nanoreactors, would reduce the polymer transfer to the organic phase. Indeed, the ICP-MS measurement of the recovered organic phases showed Rh concentrations in most cases much lower than 1 ppm, with an average of 0.24 ppm (see Table S2). There is no significant difference between the average leaching for the CCM (0.25 ppm) and NG (0.22 ppm) catalysts, whereas the measured leaching for the corresponding neutral-shell particle was much greater and architecture-dependent (1.7-2.7 ppm for the CCM [6a, c] and 0.4-1.2 ppm for the NG [7] nanoreactors).

The performance of the CCM 10% nanoreactor with a P:Rh ratio of 4:1 was further assessed in terms of catalyst recycling. All the data are collected in Table S3. In order to properly evaluate the catalyst stability and durability, the reaction time in different cycles was initially set at 2.5 h under conditions identical to those of Figure 6 and Table S2, where a fully quantitative conversion was not yet achieved. The results, shown in Figure S11, suggest the presence of a catalyst activation phase, since an essentially quantitative conversion was achieved in all runs after the first recycle. Therefore, in order to more easily detect any catalyst degradation from the conversion vs. recycle plot, a second series of recycles was carried out under the same conditions, except for setting the reaction time at 1.5 h. The corresponding results are shown in Figure 7. The presence of an initial catalyst activation phase was now even more evident, but an essentially quantitative conversion was again achieved after the 2 nd recycle and up to the 7 th recycle. Subsequently, the last two (8 th and 9 th ) recycles gave evidence for a slight decrease of final conversion. In order to verify the reproducibility of these observations, a third recycle run was carried out with a fresh catalytic charge, under the same conditions. The results (Figure S12) indeed rather faithfully reproduced those of Figure 7, including the slight decrease of final conversion after the 7 th recycle. This slight decrease may result either from mechanical losses or from catalyst degradation by adventitious oxygen diffusion during the separation of the decanted phases between subsequent cycles. From the quantitative conversions of the recycles (3-7 in Figure 7 and Figure S12), the lower limit of the catalyst TOF in this hydrogenation process is estimated as 133 h -1 . The essentially identical DLS and TEM parameters measured for the latex before catalysis and after 1 and 10 catalytic runs indicate that catalytic nanoreactors are stable (see Figure S13). DLS and TEM measurements before and after catalysis were also carried out for the NG latex, indicating again no alteration (Figure S14). In addition, the ICP-MS measurement of the Rh concentration in the recovered organic product phases gave even lower values than those indicated above (average of 0.16 ppm) and without any obvious drift for greater recycle numbers. S3, runs 11-20). The reaction time for each cycle was 1.5 h. All other conditions were identical to those of Figure 6 (styrene/1nonanol = 1:9 v/v; styrene/Rh = 200:1; T = 25°C, p(H2) = 20 bar).

c.2. Hydrogenation of neat styrene.

Since styrene is compatible with the polystyrene core and is thus able to swell the nanoreactors by itself, the hydrogenation was also tested for the neat substrate, using a styrene/Rh ratio of 5000:1. This reaction was only carried out using the CCM 10% nanoreactors, charged with the precatalysts at a P:Rh ratio of 4:1. The conversion vs. time study, see Figure 8 (data in Table S4), confirmed the presence of an initial activation phase, since only a 12.1% conversion was achieved after 5 h (average TOF = 120 h -1 ). However, the conversion was quantitative after 20 h and the slope of the conversion vs. time between 5 and 20 h yields an average TOF of ca. 300 h -1 . Nine subsequent recycles, with a reaction time of 5 h, demonstrated excellent stability. No strong evidence for an induction phase was shown in this case, probably because the catalyst is already fully activated at the end of the first cycle. For these catalytic runs, the average concentration of Rh leached into the product phase was 0.13 ppm, once again without an obvious drift for high recycle numbers. The corresponding neutral-shell CCM gave a similar TOF for the hydrogenation of neat styrene (ca. 220 h -1 ) in the first and second run, but leaching was higher (0.3-0.6 ppm) and a loss of activity was observed for the second recycle.

c.3. Hydrogenation of 1-octene in 1-nonanol and in toluene.

1-Octene is not a good solvent for polystyrene, hence its hydrogenation must be carried out in an organic solvent able to swell polystyrene. This reaction was again carried out only with the CCM 10% polymer latex and a 4:1 P:Rh ratio, using identical conditions as for the styrene/1-nonanol studies shown above (substrate concentration of 10% in volume, 25°C at 20 bar of H2 pressure). The resulting activity was quite similar to that observed for the styrene hydrogenation, leading to a nearly quantitative conversion (97.2%) after 5 h and a quantitative one after 20 h (see Table S5 and Figure S15), with octane as the only observed product and an average leaching of 0.13 ppm.

As stated above, the neutral-shell CCM led to coagulation when the hydrogenations were carried out with toluene as the carrier organic solvent. This phenomenon, due to irreversible particle coupling, was attributed to the formation of species where the Rh atoms are bonded to TPP ligands from different particle cores, which occurs because particle interpenetration is allowed by the neutral shell. In the present case, however, the charged nature of the outer shell blocks the particle interpenetration, as shown above (metal migration study). Indeed, when the hydrogenation of 1-octene catalyzed by the [RhCl(COD)]/CCM 10% latex was repeated with use of toluene as organic solvent, the decantation process was equally fast as for the corresponding reaction with 1-nonanol (see Figure S16). The substrate conversion was again quantitative (Table S5) and leaching was again very low (0.08 ppm).

Conclusion

This contribution has presented a new family of triphenylphopshine-functionalized unimolecular amphiphilic polymers with spherical morphology, with either crosslinking at the end of the hydrophobic blocks (core-crosslinked micelles, CCM) or throughout the hydrophobic core (nanogels, NG). They differ from previously described similar polymers by the nature of the outer shell, which is polycationic. These polymers have been obtained by a convergent and well-controlled RAFT polymerization and, after loading with [RhCl(COD)]2, function as efficient nanoreactors for aqueous biphasic hydrogenation of olefins. The performance of these cationic-shell nanoreactors is superior to that of the equivalent polymers with a neutral shell in terms of activity, catalyst stability, recyclability, and catalyst leaching, which can be ascribed to the greater ability of the polycationic shell to confine the nanoreactors in the aqueous phase, while not hurting the mass transport of reactants and products between the continuous organic phase and the nanoreactor core. These nanoreactors (or related ones obtained by the incorporation of other ligand-functionalized monomers) should be suitable to anchor a wide variety of catalytic metals and thus be applied to numerous other catalyzed transformations under aqueous biphasic conditions.

Experimental Section

Materials and methods. All manipulations were performed with Schlenkline techniques under an inert atmosphere of dry argon. Solvents were dried by standard procedures and distilled under argon prior to use. 4,4'-Azobis(4-cyanopentanoic acid) (ACPA, >98%, Fluka), diethylene glycol dimethacrylate (DEGDMA, 95%, Aldrich), 4-(diphenylphosphino) styrene (DPPS, 97%, Aldrich), 1-octene (>99%, ACROS Organics), n-decane (99%, Alfa Aesar), chloro(1,5-cyclooctadiene)rhodium(I) dimer ([RhCl(COD)]2, min. 40.8% Rh, ACROS Organics), 1-nonanol (>99%, TCI), 1,3,5-trioxane (>99%, Aldrich) and iodomethane (MeI, >98%, Sigma) were used as received. 4-Vinyl pyridine (4VP, 95%, Sigma) and styrene (St, 99%, Acros) were distilled under reduced pressure prior to use. The macroRAFT agent R0-(4VPMe + I -)140-b-S50-SC(S)SPr•34(DMF) (yellow powder, molar mass = 40073 g mol -1 ) was synthesized in three steps from 4-cyano-4-thiothiopropylsulfanyl pentanoic acid (CTPPA, or R0-SC(S)SPr with R0 = -C(CH3)(CN)CH2CH2COOH), [18] 4-vinyl pyridine, styrene and methyl iodide as previously described. [12] The DMF content in this product was determined by 1 H NMR in DMSO-d6 and all attempts to remove the residual 34 molecules of DMF per polymer chain were unsuccessful. Other macroRAFT agents with different number average degrees of polymerization, R0-(4VPMe + I -)x-b-S50-SC(S) SPr (x = 56, 72, 150) were prepared by the same procedure using different VP/CTPPA molar ratios. The deionized water used for the syntheses and for the DLS analyses was obtained from a Purelab Classic UV system (Elga Lab-Water). NMR spectroscopy. All nuclear magnetic resonance spectra were recorded in 5 mm diameter tubes at 297 K on a Bruker Avance 400 spectrometer. The 1 H and 31 P chemical shifts were determined using the residual peak of the deuterated solvent as internal standard and are reported in ppm (δ) relative to tetramethylsilane. Peaks are labelled as singlet (s), doublet (d), triplet (t), quadruplet (q), multiplet (m) and broad (br). For the CCM characterization, the chemical shift scale was calibrated on the basis of the solvent peak (δ 2.50 for DMSO-d6, 4.79 for D2O, 7.26 for CDCl3), and 1,3,5-trioxane (δ 5.20) was used as an integration reference. For the synthesis of the P4VP macroRAFT agent, the monomer conversion was followed by 1 H NMR spectroscopy (300 MHz Bruker) in DMSO-d6 at room temperature by the relative integration of the protons of the internal reference (1,3,5-trioxane) at 5.2 ppm and the vinylic protons of 4VP (at 5.4 and 5.9 ppm).

Characterization

Dynamic light scattering (DLS). The intensity-average diameters of the latex particles (Dz) and the polydispersity index (PDI) were obtained on a Malvern Zetasizer NanoZS equipped with a He-Ne laser (λ = 633 nm), operating at 25 °C. Samples were analyzed after dilution with deionized water, either unfiltered or after filtration through a 0.45 μm pore-size membrane. Zeta potential (ζ) measurements were also conducted on the same instrument by measuring the electrophoretic mobility.

Transmission electron microscopy (TEM). The morphological analyses of the copolymer nano-objects were performed at the Centre de Microcaractérisation Raimond Castaing (Toulouse, France) with a JEOL JEM 1400 transmission electron microscope working at 120 kV. Diluted latex samples were dropped on a formvar/carbon-coated copper grid and dried under vacuum for 24 hours. The diameter distributions of the CCM and NG particles were obtained with help of the ImageJ software, using images with 100-300 particles.

Gas chromatography (GC).

The GC test of remained substrates and products in the organic layer after catalysis was conducted by Shimadzu GC 2014 chromatograph equipped with a SLB 5ms capillary column (30 m×0.32 mm; 0.23 µm film thickness) and a flame ionization detector (FID), using helium as carrier gas. The peak assignment was assisted by a separate GC/MS analysis.

High resolution inductive couple plasmamass spectrometry (ICP-MS).

The rhodium catalyst leaching in the organic phase was quantified by high resolution ICP/MS on a XR Thermo Scientific Element. For the sample preparation, the recovered organic phase was diluted into water using a 10 4 volumetric dilution factor, high enough to ensure complete dissolution. In practice, a 100 mL volumetric flask was filled at 2/3 with Milli-Q water, then 10 µL of the organic product phase were introduced using a precision pipette. Borders were rinsed and the flask was introduced into an ultrasound bath for 15 min. The dilution was then completed with Milli-Q water to the 100 mL mark, followed by further sonication for 45 min. Standards were prepared using [RhCl(COD)]2 and triphenylphosphine dissolved in toluene, attaining Rh concentrations in aqueous solution in the 1-100 ppt range. The relative standard deviation on the measurements used for the calibration was 3%. Latexes with other a,b,c,x values (as reported in Table S1) were obtained by the same procedure from the appropriate R0-(4VPMe + I -)a-b-Sb-SC(S)SPr macroRAFT agents, using suitable molar amounts of styrene and DPPS for the chain extension. The same general procedure was used for all CCM particles and will be described in detail only for the product with a,b,c,d,x,y = 140,50,300,140,0.1,0.1 (CCM 10%). To the Schlenk tube containing the entire aqueous suspension of the R0-(4VPMe + I -)140-b-S50-b-(S0.9-co-DPPS0.1)300-SC(S)SPr (diblock 10%) polymer, prepared as described in the previous section, were successively added degassed styrene (0.702 g, 6.74 mmol, 135 equiv. per chain), DEGDMA (0.17 mL, 0.18 g, 0.75 mmol, 15 equiv. per chain), and 1.4 mL of the degassed ACPA/NaHCO3 stock solution (28.02 mg ACPA, 0.1 mmol). The resulting reaction mixture was stirred at 80 °C for 8.5 h. The monomer conversions were 91.0% for styrene and 100% for DEGDMA (by 1 H NMR in DMSO-d6).

Preparation of latexes

Cross

The final polymer has a theoretical molar mass of 93278 g mol -1 and the polymer content in the latex is 20.6 % w/w ([TPP] = 73.8 µmol/mL).

The same procedure, starting from the latex of either diblock 5% or diblock 20%, gave a latex of CCM 5% or CCM 20%, respectively. For CCM 5%: styrene (0.72 mL, 0.656 g, 6.30 mmol, 126 equiv. per chain), DEGDMA (0.16 mL, 0.17 g, 0.70 mmol, 14 equiv. per chain) and degassed ACPA/NaHCO3 stock solution (1.4 mL, 28.02 mg ACPA, 10.0 µmol); stirring at 80 °C for 4 h; conversions = 99.0% for styrene and 100% for DEGDMA; Mn,th = 90508 g mol -1 ; polymer content in the latex = 20.2 % (w/w) ([TPP] = 40.3 µmol/mL). For CCM 20%: styrene (0.72 mL, 0.655 g, 6.29 mmol, 126 equiv. per chain), DEGDMA (0.16 mL, 0.17 g, 0.75 mmol, 14 equiv. per chain) and the degassed ACPA/NaHCO3 stock solution (1.4 mL, 28.02 mg ACPA, 10.0 µmol); stirring at 80 °C for 4 h; conversions were nearly 100 % for styrene and 100% for DEGDMA; Mn,th = 98878 g mol -1 ; polymer content in the latex = 21.6 % (w/w) ([TPP] = 148.7 µmol/mL).

Latexes of CCM particles with other a,b,c,d,x,y values (as reported in Table S1) were obtained by the same procedure from the appropriate amphiphilic diblock precursors, using suitable molar amounts of styrene and DEGDMA for the crosslinking step. To the Schlenk tube containing an aqueous suspension of [R0-(4VPMe + I - )140-b-S50-SC(S)SPr]•34(DMF) polymer (0.5 g, 12.5 µmol), prepared as described previously, [12] in 6 mL of distilled water were added 1,3,5trioxane (10.6 mg, 0.12 mmol) and degassed styrene (0.62 mL, 56.2 mg, 5.39 mmol), DPPS (0.109 g, 0.38 mmol) and DEGDMA (42 µL , 45.4 mg, 187.8 µmol), and finally the degassed ACPA/NaHCO3 stock solution (0.38 mL, 7.5 mg ACPA, 27.1 µmol). The reaction mixture was stirred at 80°C for 4 h. The NMR (DMSO-d6) indicated the complete conversion of all monomers. The final polymer has a theoretical molar mass of 96732 g mol -1 and the latex has a polymer content of 16.0 % w/w ([TPP] = 53.2 µmol/mL).

General procedure for metal complexation to the phosphine ligand within polymer core. All metal complexation reactions were carried out using the same procedures, which is described here in detail for the CCM 10% polymer with a P:Rh ratio of 4:1. In a Schlenk tube was added 1 mL of the CCM 10% polymer latex (containing 73.8 µmol of TPP) and 3 mL of H2O. Toluene (3 mL) was added and the mixture was stirred for 5 min, resulting in the CCM particle core swelling. Then a separately prepared solution of [RhCl(COD)]2 (4.6 mg, 9.23 µmol) in toluene (1 mL) was added to the latex and the mixture was vigorously stirred at room temperature, stopping the stirring at regular intervals (decantation was rapid, < 1 min) to assess the reaction progress. The aqueous phase progressively became yellow while the toluene phase became completely colorless after 30 minutes of stirring. For the procedure with a P:Rh ratio of 1:1, since a slight excess of [RhCl(COD)]2 was used to ensure quantitative complexation of the TPP ligands, the resulting latex was extracted by toluene until the organic phase was colorless to remove the metal precursor excess. The measured latex volume was 5.8 mL ([TPP] = 12.7 µmol/mL).

General procedure for the aqueous biphasic catalytic hydrogenations. (a) Substrate/Rh = 200. In a vial containing a magnetic stirrer was added 1 mL of the Rh-charged latex (CCM 10%, CCM 5% or NG 10%), prepared as described in the previous sections.

The desired amount of substrate (styrene or 1-octene), mixed with 1nonanol or toluene (10% v/v), was layered on top of the latex. (b) Styrene/Rh = 5000. In a vial containing a magnetic stirrer was added 0.4 mL of CCM 10% (5.09 µmol of TPP; 1.27 µmol of Rh) and then neat styrene (0.73 mL, 664 g, 6.37 mmol). For all experiments, whatever the substrate/Rh ratio, decane (internal standard) was then added to the organic layer (substrate/decane molar ratio ≈ 4). The vial was then placed inside an autoclave, which was subsequently charged with dihydrogen (20 bar), placed in a thermostatic oil bath and stirred at 1200 rpm. At the set reaction time, the stirring was stopped, the autoclave was vented and the vial was taken out under argon. The latex decantation was rapid (< 1 min). An aliquot of the organic phase was used for the ICP-MS analysis of the Rh leaching. After phase separation, the latex was extracted with diethyl ether (3×0.3 mL). The combined organic phases were used for the GC analysis. For the recycling experiments, a fresh substrate solution (same amounts as in the initial run) was added to the same vial, followed by reaction and product separation according to the same protocol.

Figure 1 .

 1 Figure 1. Architecture and composition of the core-crosslinked micelles (CCM) and nanogel (NG) particles.

Figure 2 .

 2 Figure 2. Synthesis of the triphenylphosphine-functionalized CCM and NG particles with an outer P4VPMe + I -hydrophilic shell.

Figure 3 .

 3 Figure 3. DLS with Dz, PDI and zeta potential values (above), representative TEM images (middle) and frequency analysis of the diameters with average and standard deviation from the TEM images (below, > 100 measured particles) for: (a) CCM 5%; (b) CCM 10%; (c) CCM 20%; (d) NG. All reported DLS data were obtained on unfiltered solutions.

  Figure 4. 31 P NMR spectrum for the toluene-swollen CCM 10% latex, before (a) and after (b) equilibration with a [RhCl(COD)]2 toluene solution at a P:Rh ratio of 1:1.

  Figure 5. 31 P NMR spectra recorded at different times after mixing equivalent amounts of CCM 20% latexes with 0 and 100% Rh loadings, and stirring at room temperature: (a) 1.5 h; (b) 7 h; (c) 1 week. The starred resonance corresponds to phosphine oxide.

Figure 6 .

 6 Figure 6. Time dependence of the styrene conversion for the biphasic catalyzed styrene hydrogenation by the CCM 10% latex with a P:Rh ratio of 4:1 in 1-nonanol. Each point was generated by an independent experiment (styrene/1-nonanol = 1:9 v/v; styrene/Rh = 200:1; T = 25°C, p(H2) = 20 bar).

Figure 7 .

 7 Figure 7. Styrene conversion vs. recycle number for the biphasic catalyzed styrene hydrogenation by the CCM 10% latex with a P:Rh ratio of 4:1 in 1nonanol (data in TableS3, runs 11-20). The reaction time for each cycle was 1.5 h. All other conditions were identical to those of Figure6(styrene/1nonanol = 1:9 v/v; styrene/Rh = 200:1; T = 25°C, p(H2) = 20 bar).

Figure 8 .

 8 Figure 8. (a) Time dependence of the styrene conversion for the biphasic catalyzed hydrogenation of neat styrene by the CCM 10% latex with a P:Rh ratio of 4:1. Each point was generated by an independent experiment (styrene/Rh = 5000:1; T = 25°C, p(H2) = 20 bar). (b) Recycling under the same conditions as in (a), for t = 5 h.

  amphiphilic copolymers. The synthesis of all latexes of this type followed the same procedure, which is detailed here only for the product with a,b,c,x = 140,50,300,0.1 (diblock 10%). A stock solution of water ACPA/NaHCO3 was prepared with 5 mL of H2O and dissolution of ACPA/NaHCO3 (0.10 g / 0.10 g), [ACPA] = 71.4 mmol L -1 . The [R0-(4VPMe + I -)140-b-S50-SC(S)SPr]•34(DMF) macroRAFT agent (2 g, 49.9 μmol) was dissolved in 15 mL of degassed water under Ar in a Schlenk tube to afford a pale yellow dispersion. To this solution was added 1,3,5trioxane (11.9 mg, 0.13 mmol, used as 1 H NMR internal reference), degassed styrene(1.55 mL, 1.40 g, 13.48 mmol, 270 equiv. per chain) and DPPS (0.43 g, 1.50 mmol, 30 equiv. per chain). A portion of the degassed ACPA/NaHCO3 stock solution(1.4 mL, 28.02 mg ACPA, 0.1 mmol) was then added and the resulting reaction mixture was stirred at 80 °C for 3 h, yielding a white opalescent stable dispersion. The resulting polymer has a theoretical molar mass of 76847 g mol -1 . The weight percent polymer in the latex is 18.9%.Using the same amounts of [R0-(4VPMe + I -)140-b-S50-SC(S)SPr]•34(DMF), ACPA solution, water and trioxane but different amounts of degassed styrene and DPPS led to latexes of the product with different DPPS content (5% or 20%) in the hydrophobic block. Diblock 5%(a,b,c,x = 140,50,300,0.05): styrene(1.64 mL, 1.48 g, 14.25 mmol, 285 equiv. per chain), DPPS (0.22 g, 0.75 mmol, 15 equiv. per chain), Mn,th = 74090 g mol-1 , polymer content = 18.4% (w/w). Diblock 20% (a,b,c,x = 140,50,300,0.2): styrene(1.38 mL, 1.25 g, 13.48 mmol, 240 equiv. per chain), DPPS (0.86 g, 3.0 mmol, 60 equiv. per chain), Mn,th = 82361 g mol-1 , polymer content = 20.0 % (w/w).

  -linking of the amphiphilic copolymers. Preparation of R0-(4VPMe + I -)a-b-Sb-b-(S1-x-co-DPPSx)c-b-(S1-y-co-DEGDMAy)d-SC(S)SPr (CCM).

Rieger, F. D'Agosto, M.Lansalot and B. Charleux, Polymer Chemistry 2011, 2, 355-362.

Acknowledgements

We are grateful to the Centre National de la Recherche Scientifique (CNRS), to the Agence Nationale de la Recherche (project BIPHASNANOCAT, grant number ANR-11-BS07-025-01) and to the Institut Universitaire de France (IUF) for financial support. We are also grateful to the China Scholarship Council for a Ph.D. fellowship to HW.

Entry for the Table of Contents

Stable latexes of size-controlled spherical and unimolecular polymers, made of a crosslinked and phosphine-functionalized hydrophobic core and a poly(N-methyl-4-vinylpyridinium) shell, are efficient nanoreactors for rhodium-catalyzed hydrogenation under aqueous biphasic conditions with excellent recyclability and negligible leaching.


Chakir Bello

Yasmine Smail

Vincent Sainte-Rose

Isabelle Podglajen

Alice Gilbert

Vanessa Moreira

Fabrice Chrétien

Martine Cohen Salmon

Guy Tran Van Nhieu
email: guy.tran_van_nhieu@ens-cachan.fr

Role of astroglial Connexin 43 in pneumolysin cytotoxicity and during pneumococcal meningitis

Streptococcus pneumoniae or pneumococcus (PN) is a major causative agent of bacterial meningitis with high mortality in young infants

and elderly people worldwide. The mechanism underlying PN crossing of the blood brain barrier (BBB) and specifically, the role of non-endothelial cells of the neurovascular unit that control the BBB function, remains poorly understood. Here, we show that the astroglial connexin 43 (aCx43), a major gap junctional component expressed in astrocytes, plays a predominant role during PN meningitis. Following intravenous PN challenge, mice deficient for aCx43 developed milder symptoms and showed severely reduced bacterial counts in the brain. Immunofluorescence analysis of brain slices indicated that PN induces the aCx43dependent destruction of the network of glial fibrillary acid protein (GFAP), an intermediate filament protein specifically expressed in astrocytes and up-regulated in response to brain injury. PN also induced nuclear shrinkage in astrocytes associated with the loss of BBB integrity, bacterial translocation across endothelial vessels and replication in the brain cortex. We found that aCx4-dependent astrocyte damages could be recapitulated using in vitro cultured cells upon challenge with wild-type PN but not with a ply mutant deficient for the pore-forming toxin pneumolysin (Ply). Consistently, we showed that purified Ply requires Cx43 to promote host cell plasma membrane permeabilization in a process involving the Cx43-dependent release of extracellular ATP and prolonged increase of cytosolic Ca 2+ in host cells. These results point to a critical role for astrocytes during PN meningitis and suggest that the cytolytic activity of the major virulence factor Ply at concentrations relevant to bacterial infection requires coopting of connexin plasma membrane channels.

major PN virulence determinant Pneumolysin. Ply-mediated cytotoxicity was linked to Ca 2+ increase and required aCx43, arguing against a direct toxin activity. The results reveal a key role for astroglial signaling during PN crossing of the BBB and shed light on the mechanism of Ply-mediated cytotoxicity during meningitis. INTRODUCTION PN is a bacterial commensal of the human nasopharynx and pathogen responsible for invasive diseases, including septicaemia and meningitis with high morbidity worldwide [START_REF] Mook-Kanamori | Pathogenesis and pathophysiology of pneumococcal meningitis[END_REF][START_REF] Yau | BloodBrain Barrier Pathology and CNS Outcomes in Streptococcus pneumoniae Meningitis[END_REF]. The large majority of in vitro studies on PN translocation across the BBB have been performed using cultured brain endothelial cells that do not reflect the complexity of the BBB. Brain endothelial cells form the mechanical and highly selective barrier isolating the brain cortex from the bloodstream [START_REF] Mastorakos | The anatomy and immunology of vasculature in the central nervous system[END_REF]. The BBB selectivity is permitted by the endothelial expression of restrictive and specific transport systems, a poor pinocytic activity, and tightly sealed intercellular junctions [START_REF] Mastorakos | The anatomy and immunology of vasculature in the central nervous system[END_REF][START_REF] Abbott | Structure and function of the blood-brain barrier[END_REF]. Other cell types forming the neurovascular unit regulate the BBB function [START_REF] Abbott | Structure and function of the blood-brain barrier[END_REF][START_REF] Grab | How can microbial interactions with the blood-brain barrier modulate astroglial and neuronal function?[END_REF]. Among these, astrocytes, the most abundant cells of the Central Nervous System (CNS), form glial heterogeneous networks in various brain compartments that regulate neural and vascular functions, in particular BBB maturation and maintenance [START_REF] Alvarez | Glial influence on the blood brain barrier[END_REF][START_REF] Cabezas | Astrocytic modulation of blood brain barrier: perspectives on Parkinson's disease[END_REF][START_REF] Cohen-Salmon | Astrocytes in the regulation of cerebrovascular functions[END_REF].

Astrocytes may regulate the BBB function through signaling controlling the tightness endothelial cell junctions [START_REF] Abbott | Astrocyte-endothelial interactions at the bloodbrain barrier[END_REF]. Also, astrocytes form the glia limitans, a physical barrier limiting exchanges between the brain parenchyma and the subarachnoid space as well as between the parenchyma and parenchymal vessels [START_REF] Mastorakos | The anatomy and immunology of vasculature in the central nervous system[END_REF]. This latter barrier, also referred to as glia limitans perivascularis, consists of astrocyte processes called end-feet that entirely cover the basal membrane ensheathing the vessels of the brain parenchymal vasculature [START_REF] Abbott | Astrocyte-endothelial interactions at the bloodbrain barrier[END_REF][START_REF] Iadecola | The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease[END_REF]. At this interface, astrocytes modulate the integrity and functions of the BBB, neuroinflammation, cerebral blood flow and interstitial fluid drainage [START_REF] Iadecola | The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease[END_REF][START_REF] Aspelund | A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules[END_REF][START_REF] Boulay | Immunoregulation at the gliovascular unit in the healthy brain: A focus on Connexin 43[END_REF].

Pertinent to these functions, CNS astrocytes are highly interconnected via Connexin (Cx) channels [START_REF] Xing | Connexin Hemichannels in Astrocytes: Role in CNS Disorders[END_REF]. The Cx family consists of 21 structurally and functionally conserved proteins reported to date in mammals, designated by their apparent molecular weight [START_REF] Bock | Connexin Channels at the Glio-Vascular Interface: Gatekeepers of the Brain[END_REF]. CNS astrocytes predominantly express Cx43 and Cx30, and to a minor extent Cx26 [START_REF] Xing | Connexin Hemichannels in Astrocytes: Role in CNS Disorders[END_REF]. At gap intercellular communicating junctions, Connexins assemble into hexamers that appose to other hexamers of adjacent cells to form constitutively open channels allowing the intercellular exchange of ions and metabolites, critical for the K + and glutamate neuromodulatory buffering functions of astrocytes [START_REF] Xing | Connexin Hemichannels in Astrocytes: Role in CNS Disorders[END_REF]. Connexin hexamers also form constitutively closed hemichannels at the plasma membrane that can open upon stimulations such as increase of intracellular Ca 2+ , oxidative or mechanical stress and inflammation [START_REF] Xing | Connexin Hemichannels in Astrocytes: Role in CNS Disorders[END_REF].

Hemichannels have been shown to release ATP, glutamate, and nicotinamide adenine nucleotide (NAD) that act in a paracrine manner. In pathological conditions, massive ATP release by hemichannels and overload of intracellular Ca 2+ lead to cytotoxicity of astrocytes and other neighboring cells [START_REF] Giaume | Connexin and pannexin hemichannels in brain glial cells: properties, pharmacology, and roles[END_REF]. Despite current evidence for the role of astrocytes in the regulating the BBB, their role during its crossing by bacterial meningitis-causing microorganisms has been poorly investigated. By directly acting as a physical barrier or by indirectly controlling endothelial cell junctions, perivascular astrocytes may prevent bacterial translocation into the cortex and severe brain injury associated with encephalitis. Also, the glia limitans is believed to form a continuous structure isolating the brain parenchyma from the subarachnoid space and brain parenchymal blood capillaries [START_REF] Alvarez | Glial influence on the blood brain barrier[END_REF]. Thus, through the release of mediators in the cerebrospinal fluid (CSF) or perivascular spaces regulating endothelial cell junctions, astrocytes may regulate bacterial translocation events across meningeal or parenchymal blood vessels. During mice meningitis, PN was found to adhere to subarachnoid vessels during the first hours of infection, prior to its progressive association with brain parenchymal vessels during the 14H post-infection period [START_REF] Iovino | Interactions between blood-borne Streptococcus pneumoniae and the blood-brain barrier preceding meningitis[END_REF]. However, in these studies, PN counts in the subarachnoid space did not increase concomitantly during the infection period, questioning whether bacterial translocation across blood vessels preferentially occurred at these specific brain areas.

Numerous PN meningitis-associated factors have been reported that target host cell receptors, degrade the extracellular matrix or intercellular junctional components, trigger inflammatory responses and allow escape from innate defense mechanisms [START_REF] Mook-Kanamori | Pathogenesis and pathophysiology of pneumococcal meningitis[END_REF][START_REF] Yau | BloodBrain Barrier Pathology and CNS Outcomes in Streptococcus pneumoniae Meningitis[END_REF][START_REF] Doran | Host-pathogen interactions in bacterial meningitis[END_REF]. PN may cross the BBB via a paracellular route, following the loosening of endothelial cell junctions triggered by Ply and inflammatory responses [START_REF] Doran | Host-pathogen interactions in bacterial meningitis[END_REF]. PN can also transcytose through brain vascular endothelial cells by targeting the Platelet-Activating Factor receptor (PAFR) and the laminin receptor [START_REF] Orihuela | Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models[END_REF]. Other receptors such as the Platelet Endothelial Cell Adhesion Molecule (PECAM-1) and the poly-Immunoglobulin Receptor (pIgR) could be involved [START_REF] Yau | BloodBrain Barrier Pathology and CNS Outcomes in Streptococcus pneumoniae Meningitis[END_REF][START_REF] Iovino | Streptococcus pneumoniae Interacts with pIgR expressed by the brain microvascular endothelium but does not co-localize with PAF receptor[END_REF][START_REF] Iovino | pIgR and PECAM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion[END_REF], the latter being also targeted by PN during crossing of the nasopharyngeal epithelium [START_REF] Hammerschmidt | Adherence molecules of pathogenic pneumococci[END_REF]. Interactions between PN and cell receptors require down-regulation of the bacterial capsule, essential for bacterial survival in the blood and evasion of phagocytosis [START_REF] Yau | BloodBrain Barrier Pathology and CNS Outcomes in Streptococcus pneumoniae Meningitis[END_REF] Ply is a critical virulence factor involved in PN invasive diseases. Through various actions including cytotoxicity linked to its pore-forming toxin activity, complement activation, and binding to Toll-like Receptors, Ply was reported to promote tissue inflammation and damages, as well as high bacteremial titers [START_REF] Mitchell | The biology of pneumolysin[END_REF][START_REF] Nishimoto | Pneumolysin: Pathogenesis and Therapeutic Target[END_REF]. The role of Ply in PN crossing of the BBB is a mater of debate. Ply may favor PN translocation by triggering inflammation [START_REF] Mook-Kanamori | Characterization of a pneumococcal meningitis mouse model[END_REF][START_REF] Chen | Upregulation of CBP by PLY can cause permeability of blood-brain barrier to increase meningitis[END_REF] known to promote BBB permeabilization [START_REF] Abbott | Structure and function of the blood-brain barrier[END_REF]. However, there are conflicting reports about the role of Ply in promoting brain inflammation [START_REF] Yau | BloodBrain Barrier Pathology and CNS Outcomes in Streptococcus pneumoniae Meningitis[END_REF][START_REF] Mitchell | The biology of pneumolysin[END_REF]. Depending on studies, Ply may or may not be required for bacterial replication and meningeal inflammation [START_REF] Friedland | The limited role of pneumolysin in the pathogenesis of pneumococcal meningitis[END_REF][START_REF] Hirst | Streptococcus pneumoniae deficient in pneumolysin or autolysin has reduced virulence in meningitis[END_REF][START_REF] Mitchell | Streptococcus pneumoniae: virulence factors and variation. Clinical microbiology and infection : the official publication of the European Society of Clinical[END_REF][START_REF] Hoegen | The NLRP3 inflammasome contributes to brain injury in pneumococcal meningitis and is activated through ATP-dependent lysosomal cathepsin B release[END_REF] By interfering with complement activity, Ply is required to resist bacterial clearance to promote high bacteremial titers, therefore rendering complex the determination of its precise role during meningitis [START_REF] Kostyukova | A study of pathogenic factors of Streptococcus pneumoniae strains causing meningitis[END_REF][START_REF] Harvey | Pneumolysin with low hemolytic activity confers an early growth advantage to Streptococcus pneumoniae in the blood[END_REF][START_REF] Alcantara | Pneumolysin-induced complement depletion during experimental pneumococcal bacteremia[END_REF]. Arguing for a direct role of Ply in brain inflammation and BBB permeabilization, intravenous administration of Ply at concentrations similar to those encountered during PN meningitis leads to BBB permeabilization [START_REF] Chen | Upregulation of CBP by PLY can cause permeability of blood-brain barrier to increase meningitis[END_REF][START_REF] Hupp | Magnesium therapy improves outcome in Streptococcus pneumoniae meningitis by altering pneumolysin pore formation[END_REF]. Of note, high levels of Ply expression may antagonize with PN transcytosis accross the BBB by, and meningitis-causing PN strains express lower Ply levels than those causing sepsis [START_REF] Surve | Heterogeneity in pneumolysin expression governs the fate of Streptococcus pneumoniae during blood-brain barrier trafficking[END_REF]. Ply was shown to alter astrocyte shape, glutamate signaling and to trigger synaptic damage at non-lytic concentrations and during PN meningitis [START_REF] Iliev | Cholesterol-dependent actin remodeling via RhoA and Rac1 activation by the Streptococcus pneumoniae toxin pneumolysin[END_REF][START_REF] Fortsch | Changes in astrocyte shape induced by sublytic concentrations of the cholesterol-dependent cytolysin pneumolysin still require pore-forming capacity[END_REF][START_REF] Wippel | Bacterial cytolysin during meningitis disrupts the regulation of glutamate in the brain, leading to synaptic damage[END_REF]. However, the role of astrocytes during PN translocation across the BBB remains ill defined.

RESULTS

PN co-opts aCx43 during meningitis

Using mouse retro-orbital vein injection, we found that all aCx43 FL/FL control mice developed meningitis when challenged with TIGR4, a PN serotype 4 wild-type strain [START_REF] Yardeni | Retro-orbital injections in mice[END_REF]. All PN infected mice showed reduced activity, associated with piloerection at 9 H post-infection, with aggravating symptoms including hunched postures and absence of motility at 24 H postinfection, the longest incubation time at which mice were sacrificed to limit animal suffering (Figs 1A andS1). As shown in Fig 1B , CFU determination in brains of aCx43 FL/FL mice indicated that bacterial translocation was detected as early as 3 H post-injection and increased exponentially to reach a median value of 6.2 x 10 4 CFUs / mg after 24 H, a time point at which rupture of the BBB integrity could be detected macroscopically (Fig S1A). Bacterial CFUs could be detected in the cerebrospinal fluid at 24H but not at 3H post-infection, suggesting that PN did not translocate preferentially across meningeal vessels at early infection stages (Fig S1B). In control experiments, aCx43 FL/FL mice showed no symptoms when infected with a control non-pathogenic Escherichia coli K12 strain and no CFU counts could be detected in brains sampled at 24 H post-infection (N = 4). Interestingly, aCx43 -/- mice did not present obvious symptoms besides a slightly reduced activity up to 24 H post infection, suggesting that infection was controlled. Accordingly, PN translocation in the brain of aCx43 -/-mice was significantly decreased with a median value of 1.2 x 10 2 CFUs / mg at 24 H post-infection. aCx43 FL/FL and aCx43 -/-mice shared similar high bacteremial median values between during the first 13H incubation. However, consistent with severe sepsis, at 24 H post-infection, some aCx43 FL/FL mice showed PN blood titers that were up to 4 logs higher than titers in aCx43 -/-mice (Fig 1C). PN infection led to up-regulation of the proinflammatory cytokines TNF- and IL1-, as well as of endothelial inflammation markers as revealed by qRT-PCR, but to a similar extent in aCx43 FL/FL and aCx43 -/-mice despite the difference in bacterial titers (Fig S1C).

aCx43-dependent killing of astrocytes and loss of BBB integrity during PN translocation across the endothelial vessels

To further characterize the early events associated with PN translocation across the BBB, we performed brain immunofluorescence analysis (Material and Methods). No bacteria were detected in the brain cortex at 3 H post-infection and only 0.7 to 3.3 bacteria or bacterial clusters / mm 2 were observed at 6 H and 13 H post-infection. We did not detect preferential PN translocation in a specific brain region. At 6H post-infection, bacteria were detected in the subarachnoid space, as well as cortical and deeper parenchymal area, but not in the choroid plexus where bacteria were detected only after 13H post-infection. As shown in Fig 2A ,    however, bacteria were always found in close association with brain vessels, consistent with early brain translocation events. Immuno-labeling revealed the presence of small capsular remnants at the vicinity of bacteria, including in brain vessels and cortex, consistent with bacterial lysis or capsular shedding reported during in vitro interaction with endothelial cells also occurring in vivo (Fig 2A ,arrowhead; [START_REF] Doran | Host-pathogen interactions in bacterial meningitis[END_REF][START_REF] Kietzman | Dynamic capsule restructuring by the main pneumococcal autolysin LytA in response to the epithelium[END_REF]). Remarkably, such capsular remnants were also detected in the brain cortex seemingly leaking from vessels associated with translocated bacteria at 6H post-infection, consistent with loss of vessel integrity at early stages of crossing of the BBB by PN (Fig 2A ,arrows). Capsular remnants and loss of endothelial vessel integrity was clearly detected in association with bacterial clusters at 13H post-infection. Consistent with the scoring of CFUs from sampled brains, aCx43 -/-mice showed three times less translocation events compared to aCx43 FL/FL mice as early as 6H post-infection (Figs 2B and2C). Single or a discrete number of bacteria were observed in aCx43 -/-mice, with lesser capsular remnants and vessel destruction, consistent with poor PN translocation across the BBB (Fig 2D). Bacterial cluster size quantification also indicated that PN brain intra-cortical replication was higher in aCx43 FL/FL relative to aCx43 -/-mice, with clusters 6 and 23-times bigger at 6H and 13 H post-infection, respectively (Figs 2C and2D).

To analyze the effects of PN infection on CNS astrocytes, brain slices were immuno-labeled for the prototypical marker glial fibrillary acid protein (GFAP), the major intermediate filament protein in astrocytes. GFAP is a marker of astrocytes' activation in response to CNS injury [START_REF] Hol | Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system[END_REF]. GFAP also controls the astrocyte shape and is implicated in various functions, including their neuro-modulatory and BBB regulatory functions [START_REF] Liedtke | GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination[END_REF][START_REF] Cho | Properties of astrocytes cultured from GFAP over-expressing and GFAP mutant mice[END_REF]. Strikingly, PN translocation and intracortical growth in aCx43 FL/FL mice brains were associated with major astrocyte injury characterized by the destruction of the cytoskeletal GFAP network. GFAP debris occurred in close contact with intracortical PN microcolonies and at the vicinity defined by the area corresponding to shed capsular materials (SCMs) (Figs 3 and S2, dotted area). These observations suggested that secreted bacterial products were responsible for the cytotoxicity of astrocytes in the absence of direct bacterial contact. Furthermore, astrocytes at the vicinity of these bacterial microcolonies also showed nuclear shrinkage described for the PN pore-forming toxin Ply (Fig S3, [START_REF] Braun | Pneumolysin causes neuronal cell death through mitochondrial damage[END_REF]). In contrast, astrocyte injury as quantified by the formation of GFAP debris was not observed in aCx43 -/-mice brains (Figs 3C andS3).

Instead, rare and discrete bacteria were detected in association with intact GFAP-labeled astrocytic processes (Fig 3B).

These results indicate that PN translocation across brain vessels is associated with a local loss of BBB integrity and with aCx43-dependent astrocytic death. The occurrence of aCx43dependent astrocyte killing at a distance from intact bacteria suggests the aCx43-dependent cytotoxic action of a secreted bacterial product during PN crossing of the BBB.

Ply mediates aCx43-dependent killing of astrocytes

Ply is a well-characterized cholesterol-dependent cytolysin (CDC) forming large pores into host cell membranes that are generally thought to account for its cytotoxicity [START_REF] Mitchell | The biology of pneumolysin[END_REF]. Therefore, the involvement of aCx43 in PN-or Ply-mediated astrocyte toxicity was intriguing. To investigate this, mouse cortical primary astrocytes were isolated and challenged in vitro with PN (Material and Methods). As observed in brain slices, PN challenge for 90 min led to a clear destruction of the GFAP network associated with the formation of GFAP debris in cultured aCx43 FL/FL astrocytes following PN-challenge, which was not detected in aCx43 -/- astrocytes (Figs 4A and4B). Also consistent with in vivo results, the formation of GFAP debris was associated with nuclear shrinking and cell retraction in aCx43 FL/FL astrocytes (Figs 4A and 4C, FL+PN). Since these observations were reminiscent of cytotoxicity linked to a pore-forming toxin, we tested a PN isogenic mutant deficient for ply expression. Strikingly, nuclear shrinkage was not observed in aCx43 -/-astrocytes challenged with wild-type PN, nor in aCx43 FL/FL astrocytes challenged with the ply mutant, indicating a role for aCx43 in Ply- To further demonstrate the role of aCx43 in Ply-mediated cytotoxicity, we purified Ply in a recombinant form (Material and Methods) and used it to challenge astrocytes. As shown in Figs 3E-3G, the destruction of GFAP network and nuclear shrinkage induced by PN were phenocopied when cells were challenge with purified recombinant Ply at a concentration of 250 nM. These Ply-mediated effects were inhibited by carbenoxolone (Figs 3F and3G).

Cx43 expression confers Ply-mediated cytotoxicity linked to plasma membrane permeabilization

Our results suggested that Cx43 play a major role in Ply-mediated cytotoxicity. These findings are reminiscent of cytotoxicity induced by the small pore forming RTXs toxins involving the release of ATP through plasma membrane channels formed by purinergic P2X7 receptors and pannexins [START_REF] Skals | Alpha-hemolysin from Escherichia coli uses endogenous amplification through P2X receptor activation to induce hemolysis[END_REF][START_REF] Skals | Bacterial RTX toxins allow acute ATP release from human erythrocytes directly through the toxin pore[END_REF]. To test this, astrocytes were incubated in the presence of hexokinase to deplete ATP from the extracellular milieu [START_REF] Skals | Bacterial RTX toxins allow acute ATP release from human erythrocytes directly through the toxin pore[END_REF]. As shown in Fig 5A, Plymediated cytotoxicity was inhibited by hexokinase treatment to an extent similar to that of carbenoxolone, suggesting a role for ATP released through Cx43 hemichannels. To further confirm the implications of Cx43 in Ply-mediated cytotoxicity and extend these findings to cells other than astrocytes, we analyzed the effects of Ply in HeLa cells that do not express known Cxs and HeLa cells stably transfected with Cx43 (HCx43) [START_REF] Van Nhieu | Connexin-dependent inter-cellular communication increases invasion and dissemination of Shigella in epithelial cells[END_REF]. As shown in Figs 5B

and S4, challenge with PN did not lead to detectable change in morphology of parental HeLa cells, but cell retraction was clearly observed for HCx43 cells. As expected, cell retraction was dependent on Ply, since it was not detected upon challenge with a ply mutant and was induced by purified Ply (Figs 5B and S4). As observed for astrocytes, Ply-induced rounding of HCx43 cells was inhibited by hexokinase (Fig 4B). We next performed dye release assays to test the role of ATP release through Cx43 hemichannels in Ply-mediated plasma membrane permeabilization. As shown in Figs 5C and5D, upon challenge with Ply, the rates of calcein fluorescence decrease were significantly higher in HCx43 cells compared to parental HeLa cells indicative of higher plasma membrane permeabilization linked to Cx43 expression. Furthermore, the Cx43-dependent plasma membrane permeabilization was abrogated in the presence of hexokinase during Ply challenge, consistent with a role for ATP release through Cx43 hemichannels (Fig 5D ).

Role of Cx43 expression in Ply-mediated Ca 2+ increase

At agonist concentrations, extracellular ATP (eATP) acts in a self-feeding amplifying loop that permeabilizes plasma membranes by stimulating Ca 2+ responses that lead to the opening of hemichannels and further release of ATP in the extracellular medium [START_REF] Bock | Connexin Channels at the Glio-Vascular Interface: Gatekeepers of the Brain[END_REF][START_REF] Giaume | Connexin and pannexin hemichannels in brain glial cells: properties, pharmacology, and roles[END_REF]. Our results suggest that such a Cx43-dependent amplifying loop plays a critical role in previously described Ply-mediated cytotoxicity linked to Ca 2+ overload [START_REF] Braun | Pneumolysin causes neuronal cell death through mitochondrial damage[END_REF][START_REF] Stringaris | Neurotoxicity of pneumolysin, a major pneumococcal virulence factor, involves calcium influx and depends on activation of p38 mitogen-activated protein kinase[END_REF].

To test this, we performed Ca 2+

DISCUSSION

Our results indicate a role for aCx43 in the PN translocation across the BBB associated with a loss of brain vascular endothelial integrity and revisit the cytotoxic role of Ply at pathophysiological concentrations. aCx43, the major Cx expressed in astrocytes, forms hemichannels involved in Ca 2+ signaling particularly relevant for the regulation of the BBB permeability [START_REF] Bock | Connexin Channels at the Glio-Vascular Interface: Gatekeepers of the Brain[END_REF][START_REF] Giaume | Connexin and pannexin hemichannels in brain glial cells: properties, pharmacology, and roles[END_REF]. As a Cholesterol-Dependent Cytolysin (CDC), Ply is believed to cause cytotoxicity through the formation of large pores in host cell plasma membranes [START_REF] Mitchell | The biology of pneumolysin[END_REF][START_REF] Van Pee | CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin[END_REF]. Our findings, however, support the notion that at physiological concentrations, Ply cytotoxicity involves Cx43 hemichannels and the release of extracellular ATP to promote host cell permeabilization. Published reports show that while high concentrations, of Ply lead to plasma membrane destabilization, low concentrations induce the formation of small pores in host cell membranes, linked to the formation of incomplete rings or arcs [START_REF] El-Rachkidy | Pneumolysin generates multiple conductance pores in the membrane of nucleated cells[END_REF][START_REF] Gilbert | Measuring kinetic drivers of pneumolysin pore structure[END_REF]. Thus, our results argue that pathophysiological concentrations, Ply functions in a manner similar to small pore forming RTX toxins shown in other systems to require host cell plasma membrane channels such as Pannexins, the P2X7 purinergic receptors and ATP release to induce hemolysis [START_REF] Wang | Increased extracellular ATP: an omen of bacterial RTX toxininduced hemolysis?[END_REF]. Because aCx43 is a predominant regulator of astrocyte functions, the characterization of Cx43 as major actor in Ply-mediated cytotoxicity has important implications during PN meningitis [START_REF] Iadecola | The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease[END_REF][START_REF] Bock | Connexin Channels at the Glio-Vascular Interface: Gatekeepers of the Brain[END_REF].

Ply was previously shown to induce actin cytoskeletal alterations through Ca 2+ influx and activation of the small GTPase Rac1 [START_REF] Iliev | Cholesterol-dependent actin remodeling via RhoA and Rac1 activation by the Streptococcus pneumoniae toxin pneumolysin[END_REF]. Ply also mediates the bundling of microtubules independent of Ca 2+ influx [START_REF] Iliev | Rapid microtubule bundling and stabilization by the Streptococcus pneumoniae neurotoxin pneumolysin in a cholesterol-dependent, non-lytic and Src-kinase dependent manner inhibits intracellular trafficking[END_REF]. Here, we show that aCx43 determines the destruction of the GFAP filament network by Ply. In other studies, the destruction of GFAP filaments has been associated with activation of Ca 2+-dependent proteases [START_REF] Lee | Rapid increase in immunoreactivity to GFAP in astrocytes in vitro induced by acidic pH is mediated by calcium influx and calpain I[END_REF][START_REF] Yang | Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker[END_REF]. The increased stimulation of Ply-mediated Ca 2+ signals may therefore account to the in vivo destruction of GFAP structures linked to aCx43 expression during PN translocation in the brain. Our findings that aCx43-deficient mice are virtually resistant to PN meningitis highlight the key role of astrocytes in the regulation of the BBB integrity during infection. These findings argue for a major role of aCx43-mediated signaling in severe brain injuries linked to vascular inflammation, infarction and thrombosis of brain parenchymal vessels, often associated with death or severe neurological sequelae during PN meningitis in humans [START_REF] Engelen-Lee | Pneumococcal meningitis: clinical-pathological correlations (MeninGene-Path)[END_REF]. The results also show the aggravating role of astrocyte and aCx43-mediated signals in the PN translocation across parenchymal blood vessels. As opposed to parenchymal vessels, subarachnoid blood vessels are not ensheathed by the glia limitans. The relevance of aCx43-mediated signaling for the potential translocation of PN across meningeal vessels therefore remains to be determined. Clearly, the implications of our findings in the development of PN meningitis will require further investigation. Interestingly, Ply was also reported to be required for cardiac microlesions adjacent to blood vessels induced by PN translocation across the vascular endothelium [START_REF] Brown | Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling[END_REF]. In light of our findings, it would be interesting to determine whether Ply and Cx43-mediated signaling also play a role in the PN translocation across cardiac endothelial cells and cytotoxicity towards cardiomyocytes since Cx43 is also highly expressed in these cell types [START_REF] Márquez-Rosado | Connexin43 phosphorylation in brain, cardiac, endothelial and epithelial tissues[END_REF]. Of note, Ply was reported to induce different types of cell death, depending on its concentration, cell types or signals linked to bacterial phagocytosis [START_REF] Nishimoto | Pneumolysin: Pathogenesis and Therapeutic Target[END_REF][START_REF] Braun | Pneumolysin causes neuronal cell death through mitochondrial damage[END_REF][START_REF] Zysk | Induction of necrosis and apoptosis of neutrophil granulocytes by Streptococcus pneumoniae[END_REF][START_REF] Bewley | Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation[END_REF]]. Reminiscent of our findings, Ply-mediated necroptosis in macrophage and lung epithelial cells was linked to plasma membrane permeabilization and Ca 2+ influx [START_REF] González-Juarbe | Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia[END_REF][START_REF] González-Juarbe | Pore-forming toxin-mediated ion dysregulation leads to death receptor-independent necroptosis of lung epithelial cells during bacterial pneumonia[END_REF].

Prolonged intracellular cytosolic Ca2+ increase in response to PFTs has been described to trigger various types of cell death including apoptosis or necrosis, depending on possible costimuli, cell repair mechanisms and Ca2+ kinetics [START_REF] Bouillot | Functional Consequences of Calcium Influx Promoted by Bacterial Pore-Forming Toxins[END_REF]. Because Cx43 regulates the kinetics of Ca2+ responses induced by Ply, its expression levels and organization in hemichannels may represent an important factor determining Ply-induced cell death in different cell types.

Based on our findings, we propose that during the early stages of PN meningitis, PN triggers loss of vascular endothelial cell integrity (Fig 7). The PN-secreted Ply further favors this loss of integrity by targeting astrocytes in a process dependent on aCx43 hemichannels and eATP release ( Fig 7). Leakage from the brain vasculature may also provide with blood nutriments favoring PN intracortical growth in aCx43-proficient but not in deficient mice. Future works is required to evaluate the role of secreted PN products and hemichannel-mediated paracrine signaling at the various stages of bacterial meningitis.

MATERIAL AND METHODS

Ethics Statement

Experiments and techniques reported here complied with the ethical rules of the French agency for animal experimentation and with the Institute of Drugs, Toxicology, Chemistry, and the Environment animal ethics committee (Paris Descartes University, Agreement 86-23).

Bacterial strains, cell lines, and reagents

The ply isogenic mutant from the S. pneumoniae serotype 4 clinical isolate TIGR4 was a kind gift from Andrew Camilli (Tufts University, Boston, USA). Bacteria were grown in Todd Hewitt Broth containing (#BD249240, Thermofisher) 0.5% Yeast Extract at 37°C (#210929, Thermofisher) and plated on Columbia blood agar plates (# 43041, Biomérieux, France).

Primary astrocytes were derived from mice forebrains as previously described [START_REF] Boulay | Immune quiescence of the brain is set by astroglial connexin 43[END_REF]. HeLa cells (ATCC CCL-2) were from ATCC, and the stable HeLa cell line expressing human Cx43 were described previously [START_REF] Van Nhieu | Connexin-dependent inter-cellular communication increases invasion and dissemination of Shigella in epithelial cells[END_REF]. Cells were grown in DMEM (Dulbecco's Modified Eagle Medium, #10567-014, Thermofisher) containing 10 % fetal calf serum in a 37°C incubator supplemented with 10% CO 2 . The anesthetics Imagen (Ketamin) was from Merial and Rompun (Xylazin) was from Bayer Heathcare. The rabbit polyclonal anti-pneumococcal serotype 4 capsular antibody was from Statens Serum Institute, Copenhagen, Denmark). The mouse monoclonal anti-GFAP (Glial Fibrillary Acidic Protein) antibody (# G3893) and Alexa

Cloning and purification of recombinant Ply

Ply was cloned into the TOPO-TA cloning vector pET101D (#K10101, Thermofisher) using the following primers: 5'-CACCATGGCAAATAAAGCAGTAAATGAC-3' and 5'-GTCATTTTCTACCTTATCCTCTACCTGAGG-3'. The insert was verified by DNA sequencing. Purification of recombinant Ply was performed using Talon resin (#PT1320-1, Clontech Laboratories Inc.) affinity chromatography from freshly transformed BL21/DE3 E. coli following the manufacturer's instructions. Samples were store in 25 mM HEPES, 50 mM NaCl, 0.1% beta-mercaptoethanol in aliquots at -80°C and defrosted freshly before use.

Mouse meningitis model

aCx43 -/-mice deficient for astrogial Cx43 and respective proficient aCx43 FL/FL mice were described previously [START_REF] Boulay | Immune quiescence of the brain is set by astroglial connexin 43[END_REF]. PN cultures were freshly grown to OD 600nm = 0.2 and resuspended in PBS buffer at a final concentration of 5 x10 8 cfu/ml. Following anesthesia, 6-9 weeks old C57BL/6 mice were infected through intravenous retro-orbital injection by 50 ls of the bacterial suspension. At various time points post-infection, mice were anesthetized and blood was sampled for CFU determination. For the macroscopic analysis of the BBB integrity, mice were injected via retro-orbital vein with 60 l of PBS containing 2% Evans Blue. Mice were subjected to intracardiac perfusion with 20 mls of sterile PBS using a peristaltic pump at a flow rate of 2.5 mls / mn prior to brain sampling. Sampled brains were either flash-frozen at -80°C for subsequent RNA extraction and qRT-PCR analysis, immediately homogenized for CFU determination following plating on blood agar plates, or processed for cryosection and immunofluorescence analysis.

QRT-PCR analysis

Total RNAs were isolated from frozen brain samples homogenized in Trizol (Life Technologies) and chloroform using glass beads and the RNeasy Lipid Tissue kit (Qiagen Corp.). qRT-PCR was performed using the Superscript II reverse transcriptase kit (Invitrogen) and SYBR green PCR master kit (Applied Biosystems) and the following pairs of primers for TNF-: The primers used for VCAM, ICAM and P-Selectin were from the QuantiTect primer assay kit (Qiagen). Results are expressed following normalization using 18S RNA.

Immunofluorescence microscopy analysis

Sampled brains were embedded in OCT (Tissue-Tek, Torrance, CA) and frozen in isopentane at -25°C. 20 m sagittal sections were cut from frozen brains in a cryostat and fixed in 4%

Cell challenge with bacterial strains and pneumolysin

Cultured cells were seeded on sterile 25 mm-diameter coverslips (Deckgläser) at a density of 2 x 10 and processed for immunofluorescence staining of GFAP, F-actin, and bacterial capsule.

Calcein release assays and Ca 2+ imaging.

Calcein release assays were performed as previously described [START_REF] Guignot | The Serine Protease EspC from Enteropathogenic Escherichia coli Regulates Pore Formation and Cytotoxicity Mediated by the Type III Secretion System[END_REF]. Briefly, cultured cells were seeded on sterile 25 mm-diameter coverslips (Deckgläser) at a density of 2 x 10 5 cells / well in the day before the experiments. Cells were washed 2 times with EM buffer and loaded with calcein-AM at 3 M final concentration in EM buffer for 30 min at 21°C. Samples were washed three times with EM buffer and placed in a observation chamber on the microscope stage at 37°C. Samples were incubated with purified Ply at 300 nM final concentration and images were acquired at 470 nm excitation every 3 minutes for 60 min. The rates of calcein release were inferred from linear fits with a Pearson correlation coefficient > 0.95.

Ca 2+ imaging was performed as described previously [START_REF] Van Nhieu | Actinbased confinement of calcium responses during Shigella invasion[END_REF]. Cells were loaded with the fluorescent Ca 2+ indicator Fluo4-AM at a final concentration of 3 M for 20 min at 21°C.

Cells were washed 3 times with EM buffer and further incubated in EM buffer for 20 min prior to mounting in the observation chamber on the microscope stage. Samples were incubated with Ply at the indicated concentration. Images were acquired every 3 seconds for at least 3 min for each Ply concentration.

Image analysis

Identical acquisition and grey levels display parameters for all samples from the same set of

  mediated cytotoxicity (Fig 4C, FL+PN). Consistent with this, nuclear shrinkage was not detected in aCx43 FL/FL astrocytes in the presence of the Cx channel inhibitor carbenoxolone (Fig 4G). As expected, a Ply-deficient isogenic PN mutant did not induce any detectable nuclear shrinkage in aCx43 -/-astrocytes (Fig 4C, KO + ply).

  imaging to study the role of Cx43 at different Ply concentrations. As shown in Figs 6A and 6B, low concentrations of Ply triggered Ca 2+ responses in HCx43 cells, with ca 25% of cells showing Ca 2+ responses at Ply concentrations ranging from 1 to 125 nM, while no responses were observed in HeLa cells at these concentrations (Fig 6B). Furthermore, all HCx43 and HeLa cells showed responses at a Ply concentration of 500 nM, (Fig 6B), but Ply triggered a lasting increase in intracellular Ca 2+ that did not return to basal levels consistent with Ca 2+ overload, three-times more frequently in HCx43 cells compared to HeLa cells (Fig 6C). Together, these results indicate that Cx43-mediated signaling amplifies Ply-mediated Ca 2+ signals and favors sustained responses, consistent with Ca 2+ overload and cytotoxicity linked to plasma membrane permeabilization observed in Cx43-expressing cells.

Fluor 563 -

 563 conjugated isolectin IB4 (# I21412), secondary goat anti-mouse IgG antibody conjugated to Alexa Fluor 488 (#A11029), goat anti-rabbit IgG conjugated to Alexa Fluor 555 (A21424), and Alexa Fluor 633 Phalloidin (#A22284), Fluo4-AM calcium indicator (#F14201), calcein-AM (#C3100MP) were from Thermofisher Scientific. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, 4', 6-Diamidino-2-phenylindole dihydrochloride (DAPI, #D9542), hexokinase (#9001-51-8), carbenoxolone (# C4790) were from Sigma Aldrich.

  and 5'-CACATCGATCTGGACATGCTGT-3'; GFAP: 5'-GGGGCAAAAGCACCAAAGAAG-3' and 5'-GGGACAACTTGTATTGTGAGCC-3'.

paraformaldehyde for 15

 15 min at 21°C. Serial sections were permeabilized for 60 min in PBS containing 0.25 % Triton X-100 and 5% newborn goat serum, prior to incubation with primary antibodies at the following dilutions: anti-GFAP (1:500), anti-PN capsular (1:300), and AlexaFluor 568-IB4 (1:100). Alex-conjugated secondary antibodies and Phalloidin were used at a 1:200 dilution. DAPI was used at a 0.1 mg/ml final concentration. Samples were mounted in DAKO fluorescence mounting medium (DAKO Corp.). Fixed samples were analyzed using Eclipse Ti inverted microscopes (Nikon) equipped with a 60 x objective, a CSUX1-A1 spinning disk confocal head (Yokogawa) and a Coolsnap HQ2 camera (Roper Scientific Instruments), or a CSU1-W1 confocal head (Yokogawa) and an ORCA Flash4 CMOS camera (Hamamatsu) controlled by the Metamorph 7.7 software. For live calcein assays and Ca 2+ imaging, epifluorescence microscopy was performed using a DMRIBe microscope (LEICA microsystems) using 380 nm, 470 nm, or 546 nm LED source excitation, equipped with a Cascade 512 camera (Roper Scientific) driven by the Metamorph (7.7) software. Images were analyzed using the Metamorph software.

Fig 1 .Fig 3 .

 13 Figures legends

Fig 4 .

 4 Fig 4. Ply mediates aCx43-dependent astrocytic death.

Fig 5 .

 5 Fig 5. Cx43 expression confers Ply-mediated cytotoxicity Cells were challenged for 90 min with purified Ply (+Ply) at 250 nM or at the indicated concentration. a, wild-type asytrocytes. b-f, HeLa cells or stable transfectants expressing Cx43 (HCx43). Incubation in the presence of: +HEX: hexokinase; + CBX: 100 M Carbenoxolone. a, b, Cells were fixed and processed for the quantification of nuclear (a) or cell retraction (b). Median values are represented. > 25 cells per sample. Mann-Whitney. ****: p < 0.001. d, > 72 cells per sample. ANCOVA. ****: p < 0.001. c, d, calcein-loaded cells were incubated with 250 nM Ply. c, representative experiment of Ply-induced calcein release. d, rates of calcein decrease determined from linear fits as in "c" (Materials and Methods). Bars: median values. Mann-Whitney. ****: p < 0.001.

Fig 6 .

 6 Fig 6. Role of Cx43 in Ply-mediated Ca 2+ increase Cells were loaded with the Ca 2+ indicator Fluo-4 and Ca 2+ imaging was performed following addition of purified Ply at the indicated concentrations. a, representative traces of Ca 2+ variations in single cells. The arrows indicate the addition of Ply at the indicated concentrations. b, c, light green bars: HeLa cells; dark green bars: HCx43 cells. b, percent of cells showing Ca 2+ responses. HeLa cells; N = 3, 145 cells. HCx43 cells. N = 3, 107 cells. c, percent of cells with lasting high Ca2+ levels following treatment with 500 nM Ply. HeLa cells, N = 3, 130 cells. HCx43 cells, N = 3, 89 cells. Kruskal-Wallis. *: p = 0.049.

Fig 7 .

 7 Fig 7. Role of aCx43 in astrocyte targeting by Ply and PN meningitis.Astrocytes regulate the BBB function through their end-feet contacting blood vessels. The BBB is represented with major cell types of the vascular unit and the different endothelial,

  5 cells / well in the day before the experiments. Cells were washed 2 times with EM buffer (120mM NaCl, 7 mM KCl, 1,8 mM CaCl 2 , 0,8 mM MgCl 2 , 25 mM HEPES pH7.3)

	supplemented with 5 mM glucose. Cells were incubated with freshly grown bacteria
	resuspended in EM buffer at a final OD 600nm = 0.2, or purified Ply at the indicated
	concentrations for 90 min at 37°C. Samples were fixed with 3.7% paraformaldehyde,
	permeabilized by incubation in PBS buffer containing 0.1% Triton X-100 for 4 min at 21°C

Acknowledgments

The authors thank Philippe Mailly from the CIRB imaging facilities for help with Imaris analysis. We thank Olivera Francetic (Institut Pasteur Paris) for critical reading.