Pneumolysin, the cholesterol-dependent cytolysin of Streptococcus pneumoniae, induces inflammatory and apoptotic events in mammalian cells. Toll-like receptor 4 (TLR4) confers resistance to pneumococcal infection via its interaction with pneumolysin, but the underlying mechanisms remain to be identified. In the present study, we found that pneumolysin-induced apoptosis is also mediated by TLR4 and confers protection against invasive disease. The interaction between TLR4 and pneumolysin is direct and specific; ligand-binding studies demonstrated that pneumolysin binds to TLR4 but not to TLR2. Involvement of TLR4 in pneumolysin-induced apoptosis was demonstrated in several complementary experiments. First, macrophages from wild-type mice were significantly more prone to pneumolysin-induced apoptosis than cells from TLR4-defective mice. In gain-of-function experiments, we found that epithelial cells expressing TLR4 and stimulated with pneumolysin were more likely to undergo apoptosis than cells expressing TLR2. A specific TLR4 antagonist, B1287, reduced pneumolysin-mediated apoptosis in wild-type cells. This apoptotic response was also partially caspase dependent as preincubation of cells with the pan-caspase inhibitor zVAD-fmk reduced pneumolysin-induced apoptosis. Finally, in a mouse model of pneumococcal infection, pneumolysin-producing pneumococci elicited significantly more upper respiratory tract cell apoptosis in wild-type mice than in TLR4-defective mice, and blocking apoptosis by administration of zVAD-fmk to wild-type mice resulted in a significant increase in mortality following nasopharyngeal pneumococcal exposure. Overall, our results strongly suggest that protection against pneumococcal disease is dependent on the TLR4-mediated enhancement of pneumolysin-induced apoptosis.

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Crystals are rarely composed of a single crystal population that have grown solely from the batch of magma in which they are resident on emplacement, either by eruption or shallow intrusion. Close investigation of the majority of crystal populations reveal that they comprise up to four main components: phenocrysts, crystals co-genetic with their magmatic host; xenocrysts, crystals wholly, or in part, foreign to the magmatic host and magma system; antecrysts, crystals which are recycled one or several times before inclusion in the host magma but have an origin within the magmatic system; and microlites, which represent small co-genetic crystals which nucleate and grow rapidly on decompression and eruption. Textural analysis techniques are employed to quantify key aspects of the crystal population, including crystal shape, crystal size distributions, spatial distribution patterns and textural modification using dihedral angles. Santorini provides a case study of an active volcanic system where a combined textural analysis study has been developed, highlighting how the crystal population is being continuously modified by a series of replenishment and mixing events. Developing textural and microgeochemical techniques provides the next stage in the interrogation of crystal populations, linking textures to isotopic heterogeneities and providing fingerprints of where crystals are sourced and re-cycled.