β hemolysin/cytolysin inhibits macrophage killing activity and induction of IL-12 and NOS2 expression at sub-lytic concentrations.

<p><b>A</b>. Survival of wt (10/84) and βh/c mutant (10/84Δ<i>cylE</i>) GBS bacteria (multiplicity of infection; MOI) in primary macrophages was determined using an <i>in vitro</i> killing assay, % survival is plotted as mean ± sem of <i>n</i> = 5. <b>B</b>. Macrophage viability was assessed by MTT colorimetric assay after infection with 10/84 and 10/84Δ<i>cylE</i> bacteria at the indicated MOI, % viability is plotted as mean ± sem of 4 replicates, representative data is shown from at least 2 independent experiments. <b>C</b>. Total RNA was isolated from macrophages infected with 10/84 and 10/84Δ<i>cylE</i> bacteria (MOI: 5) at the indicated time points, and analyzed by ribonuclease protection assay (RPA) using a multi-probe template set to measure cytokine mRNA expression. Representative data from at least 3 independent experiments is shown. <b>D</b>. IL-12p40 production was measured in cell-culture supernatants from macrophages infected with 10/84 and 10/84Δ<i>cylE</i> bacteria for the indicated time points by ELISA. Data is represented as mean ± sem of 4 replicates, representative data from at least 3 independent experiments is shown. <b>E</b>. Protein extracts were prepared from macrophages infected with 10/84 and 10/84Δ<i>cylE</i> bacteria at the indicated time points and NOS2 expression measured by immunoblotting (IB), actin was used as a loading control. Representative data from at least 3 independent experiments is shown.</p

Partially purified βh/c triggers MAPK activation and inhibits IL-12 and NOS2 expression in macrophages.

<p><b>A</b>. βh/c extract was prepared from wt GBS (10/84; βh/c^e) and a mock extract from 10/84Δ<i>cylE</i> bacteria (ΔcylE^e), hemolytic activity measured by sheep red blood cell (RBC) lysis assay. <b>B</b>. Macrophages were stimulated with heat-killed 10/84Δ<i>cylE</i> bacteria (h10/84Δ, MOI: 20) in the presence of βh/c^e or ΔcylE^e at a dilution of 1∶200, and protein extracts prepared at the indicated time points. JNK and p38 activity were measured by IP kinase assay, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002812#ppat-1002812-g002" target="_blank">Fig. 2</a>. IB for p38 was used as loading control. Representative data from at least 2 independent experiments is shown. <b>C</b>. IL-12p40 was measured in cell culture supernatants by ELISA at 8 and 24 h after stimulation of macrophages with h10/84Δ in the presence of βh/c^e or ΔcylE^e. Data is represented as mean ± sem of 4 replicates, representative data from at least 3 independent experiments is shown. <b>D</b>. Protein extracts were prepared from macrophages 8 h after stimulation with h10/84Δ in the presence of βh/c^e or ΔcylE^e and NOS2 expression measured by IB, ERK expression was used as a loading control. Representative data from at least 3 independent experiments is shown.</p

Targeted deletion of p38 in macrophages increases resistance to invasive GBS infection <i>in vivo</i>.

<p><b>A</b>. p38^f/f and p38^ΔMye mice were infected i.p. with 5×10⁷ cfu GBS (10/84) and blood cfu was determined after 8 h. The cfu for individual mice is represented in the scatter plot and median indicated by the bar. Statistical analysis was performed using non-parametric Mann Whitney t-test, *p<0.05. <b>B</b>. IL-10 and IL-12p40 were measured in serum and peritoneal fluid of GBS infected mice by ELISA. Data is represented as mean ± sem, statistical analysis was performed using Mann Whitney t-test, **p<0.005. <b>C</b>. Wt mice were injected i.p. with 400 ng recombinant mouse IL-10 before i.p. infection with 5×10⁷ cfu GBS. Blood cfu was determined 8 h after infection. The cfu for individual mice is represented in the scatter plot and median indicated by the bar. Statistical analysis was performed using non-parametric Mann Whitney t-test, **p<0.005.</p

βh/c drives p38-dependent IL-10 expression in GBS-infected macrophages.

<p><b>A</b>. Primary macrophages were infected with 10/84 and 10/84Δ<i>cylE</i> (MOI 5) and total RNA prepared at the indicated time points. IL-10, IL-12p40 and NOS2 mRNA expression was measured by real-time quantitative PCR (qPCR). Data is expressed as fold induction normalized to cyclophillin mRNA expression and the mean ± sem of 3 replicates is plotted. Representative data is shown from at least 2 independent experiments. <b>B</b>. Primary macrophages from p38^f/f and p38^ΔMye mice were infected with 10/84 (MOI: 5) and protein extracts prepared at the indicated time points. JNK, p38 and IKK activity was measured by IP kinase assay, phospho-ERK (pERK) and IκBα were measured by IB, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002812#ppat-1002812-g002" target="_blank">Fig. 2</a>. <b>C</b>. Total RNA was isolated from p38^f/f and p38^ΔMye macrophages infected with 10/84 at the indicated time points and IL-10 and IL-12p40 mRNA expression was measured by qPCR. Data is expressed as fold induction normalized to cyclophillin mRNA and the mean ± sem of 3 replicates is plotted. Representative data is shown from at least 2 independent experiments.</p

βh/c-mediated JNK and p38 activation is TLR2/MyD88-independent.

<p><b>A</b>. Primary macrophages were prepared from wild type (Wt), MyD88 (Myd88) and TLR2 (Tlr2) knockout mice. Macrophages were infected with 10/84 (MOI: 5) and protein extracts prepared at the indicated time points. JNK, p38 and IKK activity were measured by IP kinase assay, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002812#ppat-1002812-g002" target="_blank">Fig. 2</a>. IB for IKKα was used as loading control. <b>B</b>. Protein extracts were prepared from Wt, Myd88−/− and Tlr2−/− macrophages infected with 10/84 and 10/84Δ<i>cylE</i> bacteria (MOI: 5) at the indicated time points and NOS2 expression measured by IB, actin was used as a loading control. Representative data from at least 2 independent experiments is shown.</p

βh/c increases JNK and p38 MAPK activation in GBS infected macrophages.

<p>Protein extracts were prepared from macrophages infected with 10/84 and 10/84Δ<i>cylE</i> bacteria at the indicated time points. JNK, p38 and IKKγ were immunoprecipitated (IP) and kinase activity was measured by incubation with recombinant substrates; GST-cJun^1–79, GST-MAPKAPK2 (GST-MK2^1–150) and GST-IκBα^1–54, in the presence of ³²P-γ-ATP. Substrates were resolved by SDS–PAGE and phosphorylation quantified by radioisotope imaging using a phosphor imager and autoradiography, fold change over control is indicated underneath the respective bands. Equal loading of kinase IP was confirmed by IB for the respective kinases. Protein extracts were also analyzed by IB for phospho-ERK, ERK and IκBα expression. Representative data from at least 3 independent experiments is shown.</p

Triggering of p38-mediated IL-10 production in macrophages is not a general property of pore-forming toxins.

<p><b>A</b>. Macrophages were stimulated with heat-killed 10/84Δ<i>cylE</i> bacteria (h10/84Δ) alone and in the presence of 1.0 µg/ml purified LLO from <i>L. monocytogenes</i>, SLO from <i>S. pyogenes</i>, or α-toxin from <i>S. aureus</i>, and protein extracts prepared at the indicated time points. JNK, p38 and IKK activity was measured by IP kinase assay, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002812#ppat-1002812-g002" target="_blank">Fig. 2</a>. Representative data from at least 2 independent experiments is shown. <b>B</b>. Viability of macrophages stimulated with h10/84Δ in the presence of 1.0 µg/ml LLO, SLO or α-toxin for 8 h was measured by MTT assay (ns: non stimulated control), data is represented as mean ± sem of 4 replicates and representative data from at least 2 independent experiments is shown. <b>C</b>. IL-12p40 and IL-10 production was measured in cell culture supernatants by ELISA at 8 and 24 h after stimulation with h10/84Δ in the presence of LLO, SLO or α-toxin. Data is represented as mean ± sem of 4 replicates and representative data from at least 2 independent experiments is shown. <b>D</b>. Protein extracts were prepared from macrophages 8 h after stimulation with h10/84Δ in the presence of βh/c^e (1∶200), LLO (0.1, 1 µg/ml), SLO (0.1, 1 µg/ml) or α-toxin (1, 2 µg/ml), and NOS2 expression measured by IB, actin was used as a loading control.</p