CD4-T-lymphocyte interactions with pneumolysin and pneumococci suggest a crucial protective role in the host response to pneumococcal infection.

Previously, we had shown that T cells accumulated in peribronchiolar and perivascular areas of lungs soon after intranasal infection with Streptococcus pneumoniae. We have now presented new evidence, using major histocompatibility class II-deficient mice, that CD4 cells are important for early protective immunity. In addition, we have also shown that a population of human CD4 cells migrates towards pneumococci and that in vivo-passaged pneumococci are substantially more potent at inducing migration than in vitro-grown bacteria. This migratory process is unique to a specific population of CD4 cells, is highly reproducible, and is independent of prior CD4 cell activation, and yet the migratory process results in a significant proportion of CD4 cells becoming activated. The production of pneumolysin is a key facet in the induction of migration of CD4 cells by in vivo bacteria, as pneumolysin-deficient bacteria do not induce migration, but the data also show that pneumolysin alone is not sufficient to explain the enhanced migration. Increased CD25 expression occurs during migration, and a higher percentage of cells in the migrated population express gamma interferon or interleukin 4 (IL-4) than in the population that did not migrate. There is evidence that the activation of IL-4 expression occurs during migration

Phage therapy is highly effective against chronic lung infections with Pseudomonas aeruginosa.

With an increase in cases of multidrug-resistant Pseudomonas aeruginosa, alternative and adjunct treatments are needed, leading to renewed interest in bacteriophage therapy. There have been few clinically relevant studies of phage therapy against chronic lung infections. Using a novel murine model that uses a natural respiratory inhalation route of infection, we show that phage therapy is an effective treatment against chronic P. aeruginosa lung infections. We also show efficacy against P. aeruginosa in a biofilm-associated cystic fibrosis lung-like environment. These studies demonstrate the potential for phage therapy in the treatment of established and recalcitrant chronic respiratory tract infections

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

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 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

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-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 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 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