HCFD feeding produces NASH in Wt mice.

<p>Wild type (Wt), myeloid IKKβ deficient (<i>IkbkbΔmye</i>), or hepatocyte IKKβ deficient (<i>IkbkbΔhep</i>) mice in both genders, were fed regular chow (Control) or high cholesterol and saturated fat diet (HCFD) from 2.5 months of age for 20 weeks. (A) Hepatomegaly was assessed by the ratio of liver weight over body weight. (B) Plasma ALT was measured. (C) Liver histological grading was determined for micro- and macro-vesicular steatosis, necroinflammation and lipogranuloma. Liver fibrosis was assessed by analysis of reticulin staining. (D) Representative microphotograph of typical NASH induced in a HCFD-fed Wt male mouse as compared to normal liver histology of a chow-fed male Wt mouse (Wt/Control, upper panels). (E) mRNA expressions in the liver of M1 genes (TNFα, MCP-1), macrophage marker CD68, IL-6, and M2 genes (Arg-1 and Ym1/Ch3l3). *p<0.05, **p<0.01, ***p<0.001 compared to Control diet within gender and within genotype. †p<0.05, ††p<0.01 compared to other genotype within gender and within diet.</p

Weight gain, visceral adiposity, and plasma glucose.

<p>Wild type (Wt), myeloid IKKβ deficient (<i>IkbkbΔmye</i>), or hepatocyte IKKβ deficient (<i>IkbkbΔhep</i>) mice in both genders, were fed regular chow (Control) or high cholesterol and saturated fat diet (HCFD) from 2.5 months of age for 20 weeks, and body weight gain (A) and visceral adiposity weight (B) were determined. Visceral adiposity was determined by summing the individual measures of epidydimal/gonadal fat, mesenteric fat, and retroperitoneal fat. Glucose tolerance (C) and insulin sensitivity (D) tests were also performed after overnight fast. *p<0.05, **p<0.01, ***p<0.001 compared to Control diet within gender and within genotype by t-test. †p<0.05, ††p<0.01 compared to other genotype within gender and within diet by ANOVA and post-hoc Tukey test.</p

Liver fibrosis induced by HCFD.

<p>Liver fibrosis as assessed by Sirius red staining (A-C, representative microphotographs), reticulin staining (D-F, representative microphotographs), or real-time PCR for α1(l)procollagen and TGFβ1 mRNA (G). Wild type (Wt), myeloid IKKβ deficient (<i>IkbkbΔmye</i>), or hepatocyte IKKβ deficient (<i>IkbkbΔhep</i>) mice were fed regular chow (Control) or high cholesterol and saturated fat diet (HCFD) from 2.5 months of age for 20 weeks (A, B, and D-F) or 12 months (C). (A, C, D, E at x169 magnification; B and F at x338 magnification.) *p<0.05, **p<0.01 compared to Control diet within gender and within genotype.</p

Upregulation of lipogenic genes and down-regulation of lipolytic genes associated with NASH induction and aggravation.

<p>Wild type (Wt), myeloid IKKβ deficient (<i>IkbkbΔmye</i>), or hepatocyte IKKβ deficient (<i>IkbkbΔhep</i>) mice in both genders, were fed regular chow (Control) or high cholesterol and saturated fat diet (HCFD) from 2.5 months of age for 20 weeks. (A) Immunoblotting of whole liver lysate and (B) densitometric analysis of pAMPK/AMPK, nSREBP-1c, PPARδ, pJNK-1/JNK-1, and pJNK-2/JNK-2. (C) Plasma adiponectin levels measured by ELISA. (D) Real-time PCR results of PPAR α, δ, and γ genes. *p<0.05, **p<0.01, ***p<0.001 compared to Control diet within gender and within genotype. †p<0.05, ††p<0.01 compared to other genotype within gender and within diet.</p

Inflammation in epididymal fat tissue induced by HCFD in Wt mice of both genders and aggravated by <i>IkbkbΔhep</i> in males.

<p>(A), (C), (E) and (G) are representative microphotographs from a male Wt mouse fed control diet from 2.5 months of age for 20 weeks as opposed to (B), (D), (F) and (H) from a male <i>IkbkbΔhep</i> mouse fed HCFD with aggravated NASH. Sections stained with H&E (x260). (B and D) Immunofluorescent staining for the macrophage marker F4/80 (red) (x260). (E and F) Immunofluorescent staining for another macrophage marker CD 68 (green) (x260). (G and H) Immunofluorescent staining for TNFα (green) (x260). (I) Fat histology scoring of inflammation in white adipose tissue was determined by assessment of inflammatory foci. (J) Real-time PCR for TNFα, Nos2, adiponectin, and the M2 gene Arg-1 in white adipose tissue. *p<0.05, **p<0.01 compared to Control diet within gender and within genotype. †p<0.05 compared to other genotype within gender and within diet.</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

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

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

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

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