Effect of capsule and pneumolysin on CXCL8 and IL-6 induction in human nasopharyngeal and bronchial epithelial cells.

<p>Detroit 562 nasopharyngeal epithelial cells (A and B) and bronchial epithelial cells (C and D) were assessed for CXCL8 (A and C) and IL-6 (B and D) release after exposure to wild type or mutant pneumococcal strains. All experiments were performed in triplicate at each of three CFU concentrations (1, 1.5 and 2 × 10⁶) and the results pooled for each strain. Note different scales of Y axes. Error bars indicate SEM. * indicates significant difference.</p

Capsule did not affect colonization of the nasopharynx but only nonencapsulated strains reached the lungs.

<p>Each symbol represents the CFU from the nasopharynx or lungs of an individual mouse on days 1, 3, 8 and 15 after intranasal inoculation. (No bacteria were detected at day 0 before any bacteria were administered.) Horizontal bars indicate means.</p

Effect of capsule and pneumolysin on CXCL8 homologue induction in the mouse nasopharynx.

<p>CXCL8 homologue (CXCL2/MIP-2) detected in nasopharyngeal homogenate of mice three days after exposure to wild type or mutant pneumococci expressed as a percentage of the value obtained with the wild type strain. Error bars indicate SEM. * indicates significant difference from value of the parent strain.</p

PLY at sub-lytic doses adversely affect cardiomyocyte function <i>in vitro</i>.

<p>(<b>A</b>) Viability of HL-1 cells was assessed 30 min after incubation with increasing concentrations of PLY and PdB using the WST-8 assay. Viability of untreated cells (UT) was set at 100%. Data are presented as Mean±SD. * <i>p</i><0.05 (n = 4). (<b>B</b>) Time course of HL-1 cell viability after incubation with 1.5 μg/ml PLY or PdB. *<i>p</i><0.05 (n = 3). (<b>C</b>) Effects of increasing concentrations of PLY on the total number of spontaneously contracting HL-1 cells over time. Data are presented as Mean±SD. *<i>p</i><0.05 (n = 4). (<b>D</b>) Representative traces of cardiomyocyte contraction before and after PLY and PdB treatment (n = 4). (<b>E-I</b>) Effects of PLY and PdB (1.0 μg/ml) on Peak Shortening (E), +dL/dt (F), TTP (G), tR₉₀ (H) and -dL/dt (I) of HL-1 cells after 30 min treatment are presented as Mean±SD. *<i>p</i><0.05 (n = 9 from 3 independent experiments).</p

Activation of PKCα-cTnI pathway and ER stress in murine cardiomyocytes exposed to D39 or PLY.

<p>(<b>A</b>) Representative Western blots and (<b>B</b>) a band-quantification histogram showing the distribution of PKCα and PKCβII in the cytosol “C” and membrane “M” fractions of murine cardiomyocytes under untreated (UT), D39- and PLN-A-infection (1x10⁶ CFU) conditions (24 h post-infection). GAPDH and Pan-Cadherin were used as markers for “C” and “M” fractions, respectively. Data are presented as Mean±SD (n = 3). * <i>p</i><0.05. (<b>C</b>) and (<b>D</b>) Typical Western blots showing the effects of D39/PLN-A (1x10⁶ CFU) infection (C) and PLY/PdB (200 ng/g) i.v. injection (D) on the association of PKCα and PKCβII with the myofilament fraction of murine cardiomyocytes 24 h post injection. cTnI was used as an endogenous control (n = 3). (<b>E</b>) and (<b>F</b>) Typical Western blots illustrating the phosphorylation of cTnI at the S43 and T144 phosphorylation sites in murine cardiomyocytes following D39/PLN-A infection (E) and PLY/PdB injection (F) (24 h post injection). cTnI was used as an endogenous control (n = 3). (<b>G</b>) and (<b>H</b>) Typical Western blots showing activation of ER stress markers in murine cardiomyocytes at 24 h post-infection with D39/PLN-A (G) and PLY/PdB (H) (n = 3).</p

PLY activates the PKCα-cTnI axis in HL-1 cardiomyocytes to depress contractility.

<p>(<b>A</b>) Representative Western blots and (<b>B</b>) a band-quantification histogram showing the distribution of PKCα and PKCβII in the cytosol “C” and membrane “M” fractions of HL-1 cells before (UT) and 30 min after PLY-treatment. GAPDH and Pan-Cadherin were used as markers for “C” and “M” fractions, respectively. Data are presented as Mean±SD (n = 3). * <i>p</i><0.05 compared to UT. (<b>C-E</b>) Typical Western blots showing PLY effects on the association of PKCα and PKCβII with the myofilament fraction (C), the phosphorylation of cTnI at the S43 and T144 PKC-dependent phosphorylation sites (D) and the effects of PKCα inhibitor Go6976 (5 nM) and the PKCβII inhibitor LY333531 (10 nM) on PLY (1.0 μg/ml)-induced phosphorylation of cTnI at S43 and T144 phosphorylation sites (E) in HL-1 cells after 30 min of PLY treatment. cTnI was used as endogenous control (n = 3). (<b>F</b>) and (<b>G</b>) Effects of PLY (1.0 μg/ml) ± Go6976 (5 nM) or LY333531 (10 nM) on Peak Shortening (F) and +dL/dt (G) after 30 min of PLY treatment. Data are presented as Mean±SD (n = 9). *<i>p</i><0.05 compared to UT.</p

PLY-expressing but not PLY-deficient pneumococci induce cardiac injury and inflammation.

<p>(<b>A-C</b>) Representative Western blots showing circulating cTnI and cTnT in murine plasma following i.v. injection of D39/PLN-A (n = 4) (1x10⁶ CFU) (A) serotype-1, sequence type 300 and 306 (n = 5) (1x10⁶ CFU) (B), and serotype 6B and PLY-deletion pneumococci (ΔPLY) (1x10⁶ CFU) (C). (<b>D</b>) Mean±SD of circulating cTnI from 4 mice (each group) that survived at 24 h after treatment with D39, PLN-A, ST300, ST306, ΔCbpA, 6B and ΔPLY (1x10⁶ CFU each). *<i>p</i><0.05 as compared to 0 h. (<b>E</b>) Linear correlation between pneumococcal CFU counts and circulating cTnI levels at various time points (n = 21). (<b>F</b>) Circulating levels of cTnI in plasma of mice following i.v. injection of D39 (1x10⁶ CFU) with or without liposomes (lipo). (n = 3 each group). *<i>p</i><0.05 as compared to 0 h, #<i>p</i><0.05 as compared to D39 group. (<b>G</b>) Survival curves of mice infected (i.v.) with D39,PLN-A and ΔPLY (n = 10 for D39, n = 5 for PLN-A, n = 4 for ΔPLY). (<b>H</b>) Circulating levels of cTnI in plasma of mice that died and those that survived within the first 30 h post-D39 infection (n = 4 each group). *<i>p</i><0.05 as compared to survival group. (<b>I</b>) Pathological examination of murine heart sections after infection with D39, serotype-1, serotype 6B and ΔCbpA pneumococci. H&E representative images (<i>a-h</i>) of murine heart sections under x4 magnification (<i>a-c</i>) and x60 magnification (<i>d-h</i>). Hearts from mice infected with D39 (30 h post infection, <i>d</i>), serotype-1 <i>(e)</i>, 6B <i>(f</i>) and ΔCbpA (<i>g</i>) show inflammatory cell infiltration (arrows) at x60 magnification, these are absent in a normal heart section (<i>h</i>). (<i>i-j</i>) Immunohistochemistry images showing absence of pneumococcal capsule staining in heart from mice infected with D39 (<i>i</i>) and serotype-1 (<i>j</i>), despite presence of inflammatory cell infiltrations (arrows). (<i>k</i>) Fresh pneumococci (D39) (arrows) were stained in parallel, as a positive control for pneumococcal capsule staining. (<i>l-n</i>) Representative immunohistochemistry images showing absence of active caspase-3 staining in heart section from mice infected with D39 (<i>l</i>), serotype-1 (<i>m</i>) and 6B (<i>n</i>) despite presence of inflammatory cell infiltrations (arrows). (<i>o</i>) Gut microvilli of a septic mouse showing positive active caspase-3 signal (arrows), was used as a positive control for active caspase-3 staining.</p

PLY binds to cardiomyocyte membrane and disrupts Ca2+ homeostasis and membrane potential at sub-lytic concentrations.

<p>(<b>A</b>) HL-1 cells were incubated with 5 μg/ml FITC-PLY and 1.0 μg/ml propidium iodide (PI) to monitor disruption of membrane integrity over time. The localisation of FITC-PLY (green) and PI (red) were recorded using time lapse confocal microscopy (LSM 710, Zeiss) in a maintained environment of 5% CO₂ at 37°C. Arrows indicate membrane binding of FITC-PLY. Bar = 20 μm (n = 3). (<b>B</b>) HL-1 cells were treated with 5, 2.5, or 1 μg/ml FITC-PLY along with 1.0 μg/ml PI for 30 min. Following washing in PBS and fixation in 4% PFA, localisation of FITC-PLY and PI were visualized under the same conditions for comparison. Typical images of each dose are presented (n = 3). Arrows indicate membrane binding of FITC-PLY. Bar = 10 μm. (<b>C</b>) HL-1 cells were incubated with 5 μg/ml FITC-PdB and 1.0 μg/ml propidium iodide (PI) to monitor disruption of membrane integrity over time. The localisation of FITC-PdB (green) and PI (red) were recorded as described in (A). Arrows indicate membrane binding of FITC-PdB. Bar = 20 μm (n = 3). (<b>D</b>) Membrane potential changes detected in HL-1 cells following exposure to PLY (1 μg/ml). Top panel: histogram showing typical resting membrane potential under untreated (UT) conditions and following exposure to PLY presented as Mean±SD (n = 3).* <i>p</i> = 0.01. Bottom panel: Typical action potential of HL-1 cells treated without (UT) or with PLY. (<b>E</b>) Changes of [Ca²⁺]_i of HL-1 cardiomyocytes with fura-2am as an indicator were recorded using the IonOptix. Typical traces before and after PLY or PdB treatment in presence and absence of extracellular Ca²⁺ are presented. Caffeine (10 mM) was used to induce Ca²⁺ release from sarcoplasmic reticulum stores within cardiomyocytes.</p

The mechanisms and effects of PLY on cardiomyocytes.

<p>High lytic concentrations of PLY induce large pore formation to lyse cells. However, <i>sub-lytic</i> concentrations of PLY bind cellular membrane to induce smaller pores thereby triggering profound Ca²⁺ influx into cardiomyocytes. The resulting abnormal increment in intracellular Ca²⁺ concentration [Ca²⁺]_i causes significant membrane depolarization, activation of detrimental signalling pathways (e.g. PKCα-cTnI axis, ER stress) and reductions in the Ca²⁺ transient amplitude to cause rhythm disturbance and depression in contractile force. Ultimately, Ca²⁺ overload causes cellular injury which may account for cardiac troponin leakage from cardiomyocytes into the circulation. Toxin-sequestering liposomes offer a potential novel therapeutic intervention against the toxic effects of circulating PLY.</p

PLY induces endoplasmic reticulum (ER) stress pathway without progressing to apoptosis in HL-1 cardiomyocytes.

<p>(<b>A</b>) Typical Western blots showing the activation of ER stress markers, (p-elF2α, p-IRE, BiP), JNK and ERK in HL-1 cells 30 min after PLY treatment. (<b>B</b>) Representative western blots showing the effect of PLY on the induction of apoptotic markers CHOP and active caspase-3 after 8 hour of treatment. Thapsigargin (Tg 5 μM) and Staurosporin (Stau 10 μM) were used as positive inducers of CHOP and active caspase-3 respectively. (<b>C-G</b>) Effects of PLY (1.0 μg/ml) and PLY+ 4-Phenylbutyric acid (4-PBA, an ER stress inhibitor, 10 mM) on Peak Shortening (C), (+dL/dt) (D), TTP (E), tR₉₀ (F) and (-dL/dt) (G) of HL-1 cells after 30 min treatment are presented as Mean±SD (n = 9 from 3 independent experiments). * <i>p</i><0.05.</p