The Evolutionarily Conserved Mediator Subunit MDT-15/MED15 Links Protective Innate Immune Responses and Xenobiotic Detoxification

Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses

Genetic diversity within and between British and Irish breeds: the maternal and paternal history of native ponies.

The UK and Ireland have many native pony breeds with historical and cultural importance as well as being a source of uncharacterized genetic diversity. However, there is a lack of comprehensive research investigating their genetic diversity and phylogenetic interrelationships. Many studies contain a limited number of pony breeds or small sample sizes for these breeds. This may result in erroneous grouping of pony breeds that otherwise have intricate interrelationships with each other and are not evaluated correctly when placed as a token subset of a larger dataset. This is the first study that specifically investigates the genetic diversity within and between British and Irish native pony breeds using large sample numbers from locations of their native origin. This study used a panel of microsatellite markers and sequence analysis of the mitochondrial control region to analyze the genetic diversity within and between 11 pony breeds from Britain and Ireland. A large dataset was collected (a total of 485 animals were used for mtDNA analysis and 450 for microsatellite analysis), and previously published data were used to place the British and Irish ponies in a global context. The native ponies of Britain and Ireland were found to have had a complex history, and the interrelationships between the breeds were revealed. Overall, high levels of genetic diversity were maintained in native breeds, although some reduction was evident in small or isolated populations (Shetland, Carneddau, and Section C). Unusual mitochondrial diversity distribution patterns were apparent for the Carneddau and Dartmoor, although among breeds and global haplogroups there was a high degree of haplotype sharing evident, well-represented within British and Irish ponies. Ancestral maternal diversity was maintained by most populations, particularly the Fells and Welsh ponies, which exhibited rare and ancient lineages. The maternal and paternal histories of the breeds are distinct, with male-biased crossings between native breeds, and other shared influences, likely Arabs and Thoroughbreds, are apparent. The data generated herein provide valuable information to guide and implement the conservation of increasingly rare native genetic resources

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Background A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. Methods This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. Findings Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. Interpretation ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials

Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway

Background: Many pathogens secrete toxins that target key host processes resulting in the activation of immune pathways. The secreted Pseudomonas aeruginosa toxin Exotoxin A (ToxA) disrupts intestinal protein synthesis, which triggers the induction of a subset of P. aeruginosa-response genes in the nematode Caenorhabditis elegans. Results: We show here that one ToxA-induced C. elegans gene, the Tribbles pseudokinase ortholog nipi-3, is essential for host survival following exposure to P. aeruginosa or ToxA. We find that NIPI-3 mediates the post-developmental expression of intestinal immune genes and proteins and primarily functions in parallel to known immune pathways, including p38 MAPK signaling. Through mutagenesis screening, we identify mutants of the bZIP C/EBP transcription factor cebp-1 that suppress the hypersusceptibility defects of nipi-3 mutants. Conclusions: NIPI-3 is a negative regulator of CEBP-1, which in turn negatively regulates protective immune mechanisms. This pathway represents a previously unknown innate immune signaling pathway in intestinal epithelial cells that is involved in the surveillance of cellular homeostasis. Because NIPI-3 and CEBP-1 are also essential for C. elegans development, NIPI-3 is analogous to other key innate immune signaling molecules such as the Toll receptors in Drosophila that have an independent role during development. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0334-6) contains supplementary material, which is available to authorized users

The Mediator subunit MDT-15 acts downstream of the p38 MAP kinase PMK-1 to regulate the induction of <i>F08G5.6</i> and <i>F35E12.5</i>.

<p>(A) Wild-type or <i>mdt-15(tm2182)</i> mutant synchronized L1 animals containing the <i>pF08G5.6::GFP</i> immune reporter were grown on vector control (L4440), <i>vhp-1</i>(RNAi) or a combination of <i>vhp-1(RNAi)</i> and <i>pmk-1(RNAi)</i> bacteria and then transferred as L4 animals to PA14 for 18 hours. Animals were photographed under the same imaging conditions. (B) qRT-PCR was used to examine the expression levels of <i>F08G5.6</i>, <i>F35E12.5</i> and <i>C32H11.1</i> in wild-type N2 and <i>mdt-15(tm2182)</i> mutant animals exposed to <i>vhp-1(RNAi)</i> or the vector control (L4440) under basal conditions (as described above) and 8 hours after exposure to <i>P. aeruginosa</i>. Knockdown of <i>vhp-1</i> caused significant induction of <i>F08G5.6</i> and <i>F35E12.5</i> in wild-type N2 animals (<i>p</i><0.001), but not in <i>mdt-15(tm2182)</i> animals (<i>p</i>>0.05), under baseline (<i>E. coli</i>) and pathogen-induced conditions. The expression of <i>C32H11.1</i> was significantly induced by <i>vhp-1(RNAi)</i> (<i>p</i><0.001) in an <i>mdt-15</i>-dependent manner under baseline conditions (<i>p</i><0.001), but not following exposure to <i>P. aeruginosa</i>. Data are the average of two biological replicates each normalized to a control gene with error bars representing SEM and are presented as the value relative to the average expression of the indicated gene in the baseline condition (L4440 animals exposed to <i>E. coli</i>).</p

MDT-15 is required for defense against <i>P. aeruginosa</i> infection.

<p>A <i>P. aeruginosa</i> pathogenesis assay with wild-type N2, <i>mdt-15(tm2182)</i> mutant worms and <i>mdt-15(tm2182)</i> animals carrying <i>pmdt-15::mdt-15</i> (three independent lines <i>agEx116</i>, <i>agEx117</i> and <i>agEx118</i>) is shown. The difference in <i>P. aeruginosa</i> susceptibility between <i>mdt-15(tm2182)</i> animals and each of the three transgenic lines carrying <i>pmdt-15::mdt-15</i> is significant, as is the survival difference between N2 and <i>mdt-15(tm2182)</i> animals (<i>p</i><0.001). For sample sizes, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004143#ppat.1004143.s009" target="_blank">Table S3</a>.</p

Protection from the toxic effects of the xenobiotic RPW-24 requires MDT-15, but not PMK-1.

<p>(A) The thirteen xenobiotic detoxification genes that were induced 4-fold or greater by RPW-24 in the NanoString nCounter gene expression analysis are presented. The top panel compares the RPW-24-mediated induction of these genes in vector control (L4440) and <i>mdt-15(RNAi)</i> animals, and the bottom panel shows these data for wild-type N2 versus <i>pmk-1(km25)</i> animals, as described in the legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004143#ppat-1004143-g002" target="_blank">Figure 2</a>. * <i>p</i><0.05 for the comparison of the RPW-24-induced conditions. (B) Vector control (L4440), <i>mdt-15(RNAi)</i> and <i>pmk-1(RNAi)</i> animals were exposed to 70 µM RPW-24 or the solvent control DMSO from the L1 stage and photographed after 70 hours of development at 20°C. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004143#ppat.1004143.s004" target="_blank">Figure S4</a> for the quantification data from this experiment.</p

The Mediator subunit MDT-15 regulates the induction of some, but not all, immune genes during <i>P. aeruginosa</i> infection.

<p>The expression of putative <i>C. elegans</i> immune effectors was analyzed by qRT-PCR in vector control (L4440) and <i>mdt-15(RNAi)</i> animals exposed to <i>P. aeruginosa</i> and the negative control <i>E. coli</i> OP50 for 8 hours. Data are the average of three replicates each normalized to a control gene with error bars representing SEM and are presented as the value relative to the average expression from all three replicates of the indicated gene in the baseline condition (L4440 animals exposed to <i>E. coli</i>). <i>mdt-15</i> expression was significantly knocked down by <i>mdt-15(RNAi)</i> in these experiments (<i>p</i><0.001).</p

Resistance to <i>P. aeruginosa</i> phenazine toxins requires MDT-15.

<p>(A) Wild-type N2, <i>mdt-15(tm2182)</i> and <i>pmk-1(km25)</i> animals were tested in the toxic phenazine <i>P. aeruginosa</i> pathogenesis assay (also called “fast killing” assay). <i>C. elegans</i> young adult animals were exposed to wild-type (WT) <i>P. aeruginosa</i>, <i>P. aeruginosa</i> carrying a deletion in both of the phenazine biosynthetic operons (<i>Δphz</i>) or <i>E. coli</i> OP50. The difference between <i>mdt-15(tm2182)</i> and N2 animals exposed to <i>P. aeruginosa</i> WT is significant (<i>p</i><0.001), as is the difference in <i>mdt-15(tm2182)</i> animals exposed to <i>P. aeruginosa</i> WT and <i>P. aeruginosa Δphz (p</i><0.001). There is no significant difference in <i>pmk-1(km25)</i> animals exposed to <i>P. aeruginosa</i> WT and <i>P. aeruginosa Δphz</i> (<i>p</i>>0.05). This assay is representative of two independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004143#ppat.1004143.s006" target="_blank">Figure S6A</a> presents the control condition for this experiment showing that both <i>C. elegans</i> N2 and <i>mdt-15(tm2182)</i> at the L4 stage are sensitive to toxic phenazine-mediated killing (<i>p</i><0.001). (B) Vector control (L4440), <i>mdt-15(RNAi)</i> and <i>pmk-1(RNAi)</i> animals were exposed to high osmolarity “fast kill” media (pH 5) with <i>E. coli</i> as the food source in the presence or absence of phenazine-1-carboxylic acid and 1-hydroxyphenazine (labeled “toxic phenazines” above) at the approximate concentrations produced by <i>P. aeruginosa</i> under standard assay conditions. The difference in killing between <i>mdt-15(RNAi)</i> animals exposed to toxic phenazines and the other conditions is significant (<i>p</i><0.001). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004143#ppat.1004143.s006" target="_blank">Figure S6</a>. For sample sizes, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004143#ppat.1004143.s009" target="_blank">Table S3</a>.</p