Heart invaded biofilm pneumococci subvert host immune response by releasing pneumolysin and rapidly kill cardiac macrophages.

<p><b>(A)</b> Western blots for pneumolysin levels in equal biomass of whole cell lysates (pellets) and supernatants of planktonic- wildtype TIGR4 (n = 3), biofilm- wildtype TIGR4 (n = 3), and planktonic T4ΩRD12 (ΩRD12) (n = 2). An isogenic pneumolysin deficient TIGR4 strain (T4 Δ<i>ply</i>) was tested as the negative control. Normalized densitometric quantification of pneumolysin levels in the supernatant is provided. Statistical analysis was performed by comparison supernatant pneumolysin levels from planktonic (PK)- wildtype TIGR4 (n = 3), and planktonic T4ΩRD12 (ΩRD12) (n = 2) to biofilm (BF)- wildtype TIGR4 (n = 3) using Welch’s <i>t</i>-test. <b>(B)</b> LDH release cytotoxicity assay of J774A.1 macrophages challenged with equal biomass of planktonic-, biofilm- TIGR4 (T4), planktonic-, biofilm- T4 Δ<i>ply</i> and planktonic-, biofilm- T4 Δ<i>ply</i> complemented with exogenous recombinant pneumolysin (rPLY, 0.3μg/mL) as determined at 0, 1, 2, 4 hours post-infection (n = 3 biological replicates, each with 3 technical replicates). Statistical analysis was performed using ordinary one-way ANOVA. <b>(C)</b> TNFα production by J774A.1 macrophages at designated time points following exposure to an equal biomass of planktonic-, biofilm- TIGR4 (T4), planktonic-, biofilm- T4 Δ<i>ply</i> and planktonic-, biofilm- T4 Δ<i>ply</i> complemented with exogenous recombinant pneumolysin (rPLY, 0.3μg/mL) as determined at 0, 1, 2, 4 hours post-infection (n = 3 biological replicates, each with 3 technical replicates). Statistical analysis was performed using ordinary one-way ANOVA. <b>(D)</b> Representative transmission electron microscopy (TEM) image of cardiac sections (magnification: 2,500X) from BALB/cJ mice infected with T4 Δ<i>ply</i> 30 hours post-infection (n = 3). <b>(E)</b> Representative high magnification immunofluorescent microscopy images of cardiac microlesions from uninfected-, passively immunized (αPly)- and naïve- mice infected with HIP or BIP 30 hours post infection, showing presence of: capsule (stained with anti-serotype 4 capsule antibody [CPS], <i>red</i>), cardiac macrophages (stained using anti-Mac-3 antibody [Mac-3], <i>green</i>), and infiltrated neutrophils (stained with anti-Ly-6G antibody [Ly-6G], <i>green</i>). A minimum of 4 stained heart sections were examined. <b>(F)</b> Absolute numbers of infiltrated neutrophils in hearts of uninfected-, passively immunized (αPly)- and naïve- mice infected with HIP or BIP 30 hours post infection. Neutrophils were identified as Gr-1⁺CD11b⁺Ly-6G⁺F4/80^- MHC-II^- cells. Statistical analysis was performed using student’s <i>t-</i>test. <b>(G)</b> Absolute numbers of cardiac macrophages in hearts of uninfected-, passively immunized (αPly)- and naïve- mice infected with HIP or BIP 30 hours post infection. Macrophages were identified as CD64⁺MerTK⁺F4/80⁺CD11b⁺ cells. Statistical analysis was performed using student’s <i>t-</i>test. <i>P</i> value: * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001; data are represented as mean ± SEM.