Laboratory Investigation of Skid Resistance for Steel Slag Utilization as Chip Seal

Slag as waste material of steel-making process has similar characteristics with aggregate that has been widely used in pavement construction. The use of slag as chip seal aggregate to provide skid resistance needs to be analyzed. In this laboratory study, the chip seal samples are made using steel slag and natural aggregate. The bonding materials used are asphalt and epoxy resin. Skid resistance tests for all chip seal samples and also hot rolled sheet pavement without chip seal application are performed using the Portable British Pendulum Tester. The results show the variations of chip seal aggregate weight are inconsistent. The natural aggregate used as chip seal material could produce high skid resistance value of 10.3% higher than that using steel slag. Also the skid resistance of chip seal with the ALD 3 mm are not significantly different with that of ALD 6 mm. Similar results occur on the skid resistance of chip seals using epoxy resin and asphalt

Histology of miR-181a1/b1^-/- (a/b KO) and control (WT).

<p><b>A</b>, Top row shows H&E staining, followed by Masson-Trichrome staining and Movat’s staining. 20X magnification was used to take the pictures. <b>B</b>, Quantification of collagen staining for all layers and vascular media between miR-181a1/b1^-/- (a/b KO) and control (WT) (n = 5–6). Whole vascular ring were used for analysis. Values are mean ±SEM, *p<0.05, **p<0.01, ***p<0.001.</p

TGF-β1 and Mut TGF- β1 3'-UTR Primers.

<p>TGF-β1 and Mut TGF- β1 3'-UTR Primers.</p

Model for the role of caspase-8 in inflammatory cytokine gene expression.

<p>TLR stimulation activates signaling complexes that mediate NF-κB and MAPK activation, which is required for induction of inflammatory cytokine gene expression. TLR stimulation also has the potential to induce programmed necrosis via RIPK3, or apoptosis via activation of caspase-8. Cleaved caspase-8 homodimers mediate apoptosis, whereas caspase-8/cFLIP heterodimers normally protect TLR-stimulated cells from programmed necrosis. Our data support a model whereby the caspase-8/cFLIP heterodimer also plays an important role in mediating inflammatory cytokine gene expression.</p

Caspase-8 regulates optimal cytokine expression independently of RIPK3 deficiency.

<p><i>B6</i>, <i>Mlkl</i>^<i>-/-</i> and <i>Mlkl</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> BMDMs were treated with LPS (100 ng/mL), Pam3CSK4 (1 μg/mL) or CpG (1 μg/mL) for 6hrs. IL-6 (A), IL-12p40 (B) and TNF (C) release was assayed by ELISA. *** <i>p</i> < 0.001 by Student's unpaired two-tailed <i>t</i>-test. Representative of two independent experiments.</p

Caspase-8 catalytic activity is required for maximal TLR-induced cytokine production.

<p>(A-F) Indicated BMDMs were pretreated with the pan-caspase inhibitor zVAD-fmk (A-C), QVD-oph (D) or IETD-fmk (F) for 1hr prior to 6 hr stimulation with LPS (100 ng/mL). TNF (F), IL12p40 (A, E, F) and IL-6 (A, E, F) were measured by ELISA, IL-1β was measured by flow cytometry (B, C) and cytotoxicity was measured by lactate dehydrogenase release (LDH) (E). All inhibitors were used at 100 μM. ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001. Student’s unpaired two-tailed <i>t</i>-test. Representative of 4 or more independent experiments. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005910#ppat.1005910.s006" target="_blank">S6 Fig</a>.</p

Caspase-8 regulates a functionally important subset of LPS-induced genes.

<p>RNA was extracted from B6, <i>Ripk3</i>^<i>-/-</i> and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> BMDMs following 6 hours of LPS treatment (100 ng/mL) and RNA-seq was performed. (A) Schematic of analysis of the data obtained from RNA-seq indicating figure panel where the results of each type of analysis is shown. (B) Principal Component Analysis (PCA) of filtered, normalized gene set displaying PC1 (95.8% of variance) against PC2 (1.5% of variance). (C) Hierarchical clustering by Pearson correlation of differentially expressed LPS-responsive caspase-8-dependent genes. Columns represent genotype and rows represent individual genes. Colored to indicate expression levels based on Z-scores. (D) GO enrichment performed in DAVID showing Biological Process terms enriched in cluster 2 from (C). Number of genes in each group are denoted above bars. Genes in cluster 1 did not show significant enrichment for any Biological Process terms. (E) Differential expression of select genes from cluster 2 and fold change (LPS-treated <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> vs LPS-treated <i>Ripk3</i>^<i>-/-</i> BMDMs). Dotted line represents 1.5-fold cutoff. (F) GSEA showing enrichment in cytokine signaling from the KEGG MSigDb canonical pathways collection 2 comparing B6 and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> LPS-treated BMDMs. GO, gene ontology; DAVID, database for annotation visualization and integrated discovery; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genome; NES, normalized enrichment score; FDR, false discovery rate. <i>R3C8</i> = <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i>. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005910#ppat.1005910.s003" target="_blank">S3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005910#ppat.1005910.s004" target="_blank">S4</a> Figs.</p

Caspase-8 self-cleavage is necessary for apoptosis but not cytokine responses.

<p>(A) Schematic of strategy used to generate <i>Casp8</i>^<i>DA/DA</i> mice using CRISPR/Cas9. GuideRNA is in blue. (B) <i>Casp8</i>^<i>+/+</i>, <i>Casp8</i>^<i>DA/+</i>, <i>Casp8</i>^<i>DA/DA</i> and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> BMDMs were infected with YopJ-deficient (ΔYopJ) and wild type <i>Yersinia</i> and caspase-8 processing was measured by western analysis. (C) BMDMs were pretreated with GSK’ 872 or vehicle control 1 hr prior to infection with <i>Yersinia</i>. Cytotoxicity was measured by LDH release 4 hrs post-infection. (D) Cleaved caspase-3 was measured by flow cytometry in BMDMs 2 hrs post-indicated treatments. (E-G) <i>Casp8</i>^<i>+/+</i>, <i>Casp8</i>^<i>DA/+</i>, <i>Casp8</i>^<i>DA/DA</i> and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> BMDMs were treated with PAMPs and cytokine production was measured after 6 hrs by flow cytometry. Representative flow plots of IL-12p40 production in response to LPS (100 ng/mL) (E), quantification of percentage of IL-12p40⁺ in response to LPS (100 ng/mL), Pam3CSK4 (1 μg/mL) or CpG (1 μg/mL) (F), IL-1β⁺ and IL-6⁺ in response to LPS (100 ng/mL) (G) (H) BMDMs were treated with LPS (100 ng/mL), Pam3CSK4 (1 μg/mL) or CpG (1 μg/mL) for 6 hrs and TNF production was measured by ELISA. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001. Student’s unpaired two tailed <i>t</i>-test. Representative of 3–5 independent experiments.</p

<i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> bone marrow-derived macrophages are defective in both MyD88 and TRIF-dependent cytokine production.

<p>(A) Bone marrow-derived macrophages (BMDMs) from B6, <i>Ripk3</i>^<i>-/-</i> and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> were infected with <i>Yersinia</i> or <i>Salmonella</i> for 24hrs, and IL-6 and IL-12p40 cytokine levels present in supernatants were quantified by ELISA. (B) IL-6, IL-12p40 and TNF production was measured by ELISA from the indicated BMDMs treated with LPS (100 ng/mL) for 6hrs. (C) IL-1β expression was measured by flow cytometry from the indicated BMDMs treated with LPS (100 ng/mL) for 5hrs. (D) BMDMs were treated with LPS (100 ng/mL) and <i>Ifnb</i> mRNA was assayed by RT-qPCR at the indicated time points. (E) Resident peritoneal macrophages from indicated mouse genotypes were isolated and stimulated <i>ex vivo</i> with LPS (10 ng/mL) for 4 hrs. TNF production by large peritoneal macrophages (LPMs) was measured by flow cytometry. (F) IL-6, IL-12p40 and TNF production was measured by ELISA from BMDMs treated with Poly(I:C) (50 μg/mL) for 24 hrs, CpG (1 μg/mL) or Pam3CSK4 (1 μg/mL) for 6hrs.(G) BMDMs were treated with LPS (100 ng/mL) and <i>Il1b</i>, <i>Il12b</i> and <i>Il6</i> mRNA was assayed by RT-qPCR at the indicated time points. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 by Student's unpaired two-tailed <i>t</i>-test. Representative of minimum of three independently performed experiments with triplicate samples for each condition. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005910#ppat.1005910.s002" target="_blank">S2 Fig</a>.</p

<i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> inflammatory monocytes and neutrophils have a cell-intrinsic defect in IL-6 and TNF production.

<p>(A) Schematic of mixed bone marrow chimera experimental set-up. Congenically marked B6 (black), <i>Ripk3</i>^<i>-/-</i> (green) or <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> (orange) bone marrow (BM) were injected at a 1:1 ratio into lethally-irradiated recipient B6.SJL mice. 8 weeks after reconstitution, chimeras were orally infected with <i>Yersinia</i> (1x10⁸/mouse) and immune responses were assayed at day 5 post-infection. (B) Quantification of percentage of Ly6C^hi inflammatory monocytes that express TNF or IL-6 for each genotype of cells in the B6:<i>Ripk3</i>^<i>-/-</i> (B6:<i>R3</i>^<i>-/-</i>), <i>Ripk3</i>^<i>-/-</i>:<i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> (<i>R3</i>^<i>-/-</i>:<i>R3</i>^<i>-/-</i><i>C8</i>^<i>-/-</i>) and B6:<i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> (B6:<i>R3</i>^<i>-/-</i><i>C8</i>^<i>-/-</i>) mixed chimeras, as indicated. Color scheme of bars is as in (A) with black bars representing B6, green bars representing <i>Ripk3</i>^<i>-/-</i>, and orange bars representing <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> cells. (C) Representative flow plots of TNF (top row of plots) and IL-6 (bottom row of plots) production in inflammatory monocytes from mixed chimeras in (B). Flow plots within each set of brackets represent cells analyzed from the same mixed bone marrow recipient mouse; genotypes of the cells analyzed are indicated above each plot <i>R3</i>^<i>-/-</i>–<i>Ripk3</i>^<i>-/-</i>, <i>R3</i>^<i>-/-</i><i>C8</i>^<i>-/-</i>—<i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i>. (D) Quantification of mean fluorescence intensity (MFI) of TNF⁺ and IL-6⁺ inflammatory monocytes from B6:<i>R3</i>^<i>-/-</i>, <i>R3</i>^<i>-/-</i>:<i>R3</i>^<i>-/-</i><i>C8</i>^<i>-/-</i>and B6:<i>R3</i>^<i>-/-</i><i>C8</i>^<i>-/-</i> mixed chimeras in (B) and (C). (E) Representative flow plots of TNF production in Ly6G⁺ neutrophils from B6:<i>R3</i>^<i>-/-</i>, <i>R3</i>^<i>-/-</i>:<i>R3</i>^<i>-/-</i><i>C8</i>^<i>-/-</i> and B6:<i>R3</i>^<i>-/-</i><i>C8</i>^<i>-/-</i> mixed chimeras analyzed as described in (C). (F) Quantification of percentage of TNF⁺ neutrophils from (E). (G) Quantification of bacterial burden per gram tissue (CFU/g). Solid bars indicate geometric mean of samples. Dotted lines indicate the limit of detection. Gating strategy is described in detail in Materials and Methods. Data are representative of 4 independently performed <i>Yersinia</i> infection experiments using 6–7 mice per experimental group. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 by Student’s unpaired two-tailed <i>t</i>-test. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005910#ppat.1005910.s001" target="_blank">S1 Fig</a>.</p