An Environmental Study and Design of River Access Sites Along the Historic Blackstone River Corridor in Rhode Island

The Blackstone River once was integral to life and industry to many towns in northern Rhode Island, but industrial pollution caused it to become unusable. As environmental remediation continues to improve the river, the Site Feasibility Access Project focuses on assessing the environmental risk of five potential river access sites located along the Blackstone River in Pawtucket, Central Falls, North Smithfield, and Cumberland; determining which site is ideal for development, and formulating river access designs for river rescue. The Blackstone Environmental Service Team (BEST) completed Corridor Land Use Evaluations (CLUE) for the sites by inspecting each site and collecting historical data and information from Government Offices and the Rhode Island Historical Society. Each site was rated using the CLUE rating system developed by BEST and a feasible site was selected based on those results. Research on river accessibility was conducted and Rhode Island state standards and specifications were collected. Two river access designs were developed for the Pawtucket Water Supply Board site: one based on current land use conditions and one to integrate with the proposed Blackstone River Bikeway. The initial results suggest that this process of Corridor Land Use Evaluations and river access design can be used in future developments along the river for safety, recreation and limited commercial purposes

Activity of Uncleaved Caspase-8 Controls Anti-bacterial Immune Defense and TLR-Induced Cytokine Production Independent of Cell Death.

Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense

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