Ribonuclease H2, RNA:DNA hybrids and innate immunity

The activation of the innate immune system is the first line of host defence against infection. Nucleic acids can potently stimulate this response and trigger a series of signalling cascades leading to cytokine production and the establishment of an inflammatory state. Mutations in genes encoding nucleases have been identified in patients with autoimmune diseases, including Aicardi-Goutières syndrome (AGS). This rare childhood inflammatory disorder is characterised by the presence of high levels of the antiviral cytokine interferon-α in the cerebrospinal fluid and blood, which is thought to be produced as a consequence of the activation of the innate immunity by unprocessed self-nucleic acids. This thesis therefore aimed to define the role of one of the AGS nucleases, the Ribonuclease H2 (RNase H2) complex, in innate immunity, and to establish if nucleic acid substrates of this enzyme were able to induce type I interferon production in vitro. The AGS nucleases may function as components of the innate immune response to nucleic acids. Consistent with this hypothesis, RNase H2 was constitutively expressed in immune cells, however, its expression was not upregulated in response to type I interferons. RNase H2-deficient cells responded normally to a range of nucleic acid PAMPs, which implied that a role for RNase H2 as a negative regulator of the immune response was unlikely, in contrast to the reported cellular functions of two other AGS proteins, TREX1 and SAMHD1. Therefore, no clear evidence was found for the direct involvement of RNase H2 in the innate immune response to nucleic acids. An alternative model for the pathogenesis of disease hypothesises that decreased RNase H2 activity within the cell results in an accumulation of RNA:DNA hybrids. To investigate the immunostimulatory potential of such substrates, RNA:DNA hybrids with different physiochemical properties were designed and synthesised. Methods to purify the hybrids from other contaminating nucleic acid species were established and their capacity as activators of the innate immune response tested using a range of in vitro cellular systems. A GU-rich 60 bp RNA:DNA hybrid was shown to be an effective activator of a pro-inflammatory cytokine response exclusively in Flt3-L bone marrow cultures. This response was completely dependent on signalling involving MyD88 and/or Trif, however the specific receptor involved remains to be determined. Reduced cellular RNase H2 activity did not affect the ability of Flt3-L cultures to mount a cytokine response against the RNA:DNA hybrid. These in vitro studies suggested that RNA:DNA hybrids may be a novel nucleic acid PAMP. Taken together, the data in this thesis suggest that the cellular function of RNase H2 is in the suppression of substrate formation rather than as a component of the immune response pathways. Future studies to identify endogenous immunostimulatory RNA:DNA hybrids and the signalling pathways activated by them should provide a detailed understanding of the molecular mechanisms involved in the pathogenesis of AGS and related autoimmune diseases

Type 2 Diabetes and Memory: Using Neuroimaging to Understand the Mechanisms

The most robust and frequently reported cognitive deficits in type 2 diabetes (DM2) are those that relate to memory. Behavioural research has identified a number of potential contributory physiological factors, including abnormalities in glucose metabolism, such as hyperglycaemia and hypoglycaemia. The impact of these mechanisms on memory has been further investigated through the use of both structural and functional neuroimaging. Structural brain imaging has indicated that memory impairments in DM2 are associated with global atrophy of the brain. Further data suggest that localised atrophy in the hippocampal area, a brain region critical to memory formation and consolidation, may be primarily responsible for the memory deficits seen in this population. Functional imaging data has corroborates these findings, with functional magnetic resonance imaging (fMRI) suggesting reduced connectivity between the hippocampus and surrounding brain regions, particularly the frontal and temporal gyri. Despite this, little functional neuroimaging research has directly investigated differences in regional brain activity between healthy and DM2 participants whilst memory tasks are being performed. By using neuroimaging techniques to their full potential, we can acquire a fuller, more comprehensive picture of the impact that DM2 has on memory

Lens Model and Time Delay Predictions for the Sextuply Lensed Quasar SDSS J2222+2745

SDSS J2222+2745 is a galaxy cluster at z=0.49, strongly lensing a quasar at z=2.805 into six widely separated images. In recent HST imaging of the field, we identify additional multiply lensed galaxies, and confirm the sixth quasar image that was identified by Dahle et al. (2013). We used the Gemini North telescope to measure a spectroscopic redshift of z=4.56 of one of the secondary lensed galaxies. These data are used to refine the lens model of SDSS J2222+2745, compute the time delay and magnifications of the lensed quasar images, and reconstruct the source image of the quasar host and a second lensed galaxy at z=2.3. This second galaxy also appears in absorption in our Gemini spectra of the lensed quasar, at a projected distance of 34 kpc. Our model is in agreement with the recent time delay measurements of Dahle et al. (2015), who found tAB=47.7+/-6.0 days and tAC=-722+/-24 days. We use the observed time delays to further constrain the model, and find that the model-predicted time delays of the three faint images of the quasar are tAD=502+/-68 days, tAE=611+/-75 days, and tAF=415+/-72 days. We have initiated a follow-up campaign to measure these time delays with Gemini North. Finally, we present initial results from an X-ray monitoring program with Swift, indicating the presence of hard X-ray emission from the lensed quasar, as well as extended X-ray emission from the cluster itself, which is consistent with the lensing mass measurement and the cluster velocity dispersion.Comment: 16 pages, 11 figures; submitted to Ap

Infection with a Brazilian isolate of Zika virus generates RIG‐I stimulatory RNA and the viral NS5 protein blocks type I IFN induction and signalling

Zika virus (ZIKV) is a major public health concern in the Americas. We report that ZIKV infection and RNA extracted from ZIKV infected cells potently activated the induction of type I interferons (IFNs). This effect was fully dependent on the mitochondrial antiviral signalling protein (MAVS), implicating RIG‐I‐like receptors (RLRs) as upstream sensors of viral RNA. Indeed, RIG‐I and the related RNA sensor MDA5 contributed to type I IFN induction in response to RNA from infected cells. We found that ZIKV NS5 from a recent Brazilian isolate blocked type I IFN induction downstream of RLRs and also inhibited type I IFN receptor (IFNAR) signalling. We defined the ZIKV NS5 nuclear localization signal and report that NS5 nuclear localization was not required for inhibition of signalling downstream of IFNAR. Mechanistically, NS5 blocked IFNAR signalling by both leading to reduced levels of STAT2 and by blocking phosphorylation of STAT1, two transcription factors activated by type I IFNs. Taken together, our observations suggest that ZIKV infection induces a type I IFN response via RLRs and that ZIKV interferes with this response by blocking signalling downstream of RLRs and IFNAR

Spatially Resolved Patchy Lyman-α\alpha Emission Within the Central Kiloparsec of a Strongly Lensed Quasar Host Galaxy at z = 2.8

We report the detection of extended Lyman-$\alpha$ emission from the host galaxy of SDSS~J2222+2745, a strongly lensed quasar at $z = 2.8$. Spectroscopic follow-up clearly reveals extended Lyman-$\alpha$ in emission between two images of the central active galactic nucleus (AGN). We reconstruct the lensed quasar host galaxy in the source plane by applying a strong lens model to HST imaging, and resolve spatial scales as small as $\sim$200 parsecs. In the source plane we recover the host galaxy morphology to within a few hundred parsecs of the central AGN, and map the extended Lyman-$\alpha$ emission to its physical origin on one side of the host galaxy at radii $\sim$0.5-2 kpc from the central AGN. There are clear morphological differences between the Lyman-$\alpha$ and rest-frame ultraviolet stellar continuum emission from the quasar host galaxy. Furthermore, the relative velocity profiles of quasar Lyman-$\alpha$, host galaxy Lyman-$\alpha$, and metal lines in outflowing gas reveal differences in the absorbing material affecting the AGN and host galaxy. These data indicate the presence of patchy local intervening gas in front of the central quasar and its host galaxy. This interpretation is consistent with the central luminous quasar being obscured across a substantial fraction of its surrounding solid angle, resulting in strong anisotropy in the exposure of the host galaxy to ionizing radiation from the AGN. This work demonstrates the power of strong lensing-assisted studies to probe spatial scales that are currently inaccessible by other means.Comment: Accepted to ApJ Letters; 7 pages, 5 figure

Strong Lens Models for 37 Clusters of Galaxies from the SDSS Giant Arcs Survey

We present strong gravitational lensing models for 37 galaxy clusters from the SDSS Giant Arcs Survey. We combine data from multi-band Hubble Space Telescope WFC3imaging, with ground-based imaging and spectroscopy from Magellan, Gemini, APO, and MMT, in order to detect and spectroscopically confirm new multiply-lensed background sources behind the clusters. We report spectroscopic or photometric redshifts of sources in these fields, including cluster galaxies and background sources. Based on all available lensing evidence, we construct and present strong lensing mass models for these galaxy clusters.Comment: 53 pages; submitted to ApJ

Increasing the proportion of healthier foods available with and without reducing portion sizes and energy purchased in worksite cafeterias:protocol for a stepped-wedge randomised controlled trial

BACKGROUND: Overconsumption of energy from food contributes to high rates of overweight and obesity in many populations. A promising set of interventions tested in pilot studies in worksite cafeterias, suggests energy intake may be reduced by increasing the proportion of healthier - i.e. lower energy - food options available, and decreasing portion sizes. The current study aims to assess the impact on energy purchased of i. increasing the proportion of lower energy options available; ii. combining this with reducing portion sizes, in a full trial. METHODS: A stepped-wedge randomised controlled trial in 19 worksite cafeterias, where the proportion of lower energy options available in targeted food categories (including main meals, snacks, and cold drinks) will be increased; and combined with reduced portion sizes. The primary outcome is total energy (kcal) purchased from targeted food categories using a pooled estimate across all sites. Follow-up analyses will test whether the impact on energy purchased varies according to the extent of intervention implementation. DISCUSSION: This study will provide the most reliable estimate to date of the effect sizes of two promising interventions for reducing energy purchased in worksite cafeterias. TRIAL REGISTRATION: The study was prospectively registered on ISRCTN (date: 24.05.19; TRN: ISRCTN87225572; doi: https://doi.org/10.1186/ISRCTN87225572)

PA-X antagonises MAVS-dependent accumulation of early type I interferon messenger RNAs during influenza A virus infection

The sensing of viral nucleic acids by the innate immune system activates a potent antiviral response in the infected cell, a key component of which is the expression of genes encoding type I interferons (IFNs). Many viruses counteract this response by blocking the activation of host nucleic acid sensors. The evolutionarily conserved influenza A virus (IAV) protein PA-X has been implicated in suppressing the host response to infection, including the expression of type I IFNs. Here, we characterise this further using a PA-X-deficient virus of the mouse-adapted PR8 strain to study activation of the innate immune response in a mouse model of the early response to viral infection. We show that levels of Ifna4 and Ifnb1 mRNAs in the lungs of infected mice were elevated in the absence of PA-X and that this was completely dependent on MAVS. This therefore suggests a role for PA-X in preventing the accumulation of early type I IFN mRNAs in the lung during IAV infection

RNA:DNA hybrids are a novel molecular pattern sensed by TLR9.

The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen-associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral-derived sequences efficiently induce pro-inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88-dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease