Termination of STING responses is mediated via ESCRT-dependent degradation

Published online 4 May 2023cGAS-STING signalling is induced by detection of foreign or mislocalised host double-stranded (ds)DNA within the cytosol. STING acts as the major signalling hub, where it controls production of type I interferons and inflammatory cytokines. Basally, STING resides on the ER membrane. Following activation STING traffics to the Golgi to initiate downstream signalling and subsequently to endolysosomal compartments for degradation and termination of signalling. While STING is known to be degraded within lysosomes, the mechanisms controlling its delivery remain poorly defined. Here we utilised a proteomics-based approach to assess phosphorylation changes in primary murine macrophages following STING activation. This identified numerous phosphorylation events in proteins involved in intracellular and vesicular transport. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We subsequently identified that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on vesicles, which facilitates the degradation of STING in murine macrophages. Disruption of ESCRT functionality greatly enhanced STING signalling and cytokine production, thus characterising a mechanism controlling effective termination of STING signalling.Katherine R Balka, Rajan Venkatraman, Tahnee L Saunders, Angus Shoppee, Ee Shan Pang, Zoe Magill, Jihane Homman-Ludiye, Cheng Huang, Rachael M Lane, Harrison M York, Peck Tan, Ralf B Schittenhelm, Senthil Arumugam, Benjamin T Kile, Meredith O, Keeffe, Dominic De Nard

Neutralization-guided design of HIV-1 envelope trimers with high affinity for the unmutated common ancestor of CH235 lineage CD4bs broadly neutralizing antibodies

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: A systematic analysis for the Global Burden of Disease Study 2015

Background: The Global Burden of Diseases, Injuries, and Risk Factors Study 2015 provides an up-to-date synthesis of the evidence for risk factor exposure and the attributable burden of disease. By providing national and subnational assessments spanning the past 25 years, this study can inform debates on the importance of addressing risks in context. Methods: We used the comparative risk assessment framework developed for previous iterations of the Global Burden of Disease Study to estimate attributable deaths, disability-adjusted life-years (DALYs), and trends in exposure by age group, sex, year, and geography for 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks from 1990 to 2015. This study included 388 risk-outcome pairs that met World Cancer Research Fund-defined criteria for convincing or probable evidence. We extracted relative risk and exposure estimates from randomised controlled trials, cohorts, pooled cohorts, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. We developed a metric that allows comparisons of exposure across risk factors—the summary exposure value. Using the counterfactual scenario of theoretical minimum risk level, we estimated the portion of deaths and DALYs that could be attributed to a given risk. We decomposed trends in attributable burden into contributions from population growth, population age structure, risk exposure, and risk-deleted cause-specific DALY rates. We characterised risk exposure in relation to a Socio-demographic Index (SDI). Findings: Between 1990 and 2015, global exposure to unsafe sanitation, household air pollution, childhood underweight, childhood stunting, and smoking each decreased by more than 25%. Global exposure for several occupational risks, high body-mass index (BMI), and drug use increased by more than 25% over the same period. All risks jointly evaluated in 2015 accounted for 57·8% (95% CI 56·6–58·8) of global deaths and 41·2% (39·8–42·8) of DALYs. In 2015, the ten largest contributors to global DALYs among Level 3 risks were high systolic blood pressure (211·8 million [192·7 million to 231·1 million] global DALYs), smoking (148·6 million [134·2 million to 163·1 million]), high fasting plasma glucose (143·1 million [125·1 million to 163·5 million]), high BMI (120·1 million [83·8 million to 158·4 million]), childhood undernutrition (113·3 million [103·9 million to 123·4 million]), ambient particulate matter (103·1 million [90·8 million to 115·1 million]), high total cholesterol (88·7 million [74·6 million to 105·7 million]), household air pollution (85·6 million [66·7 million to 106·1 million]), alcohol use (85·0 million [77·2 million to 93·0 million]), and diets high in sodium (83·0 million [49·3 million to 127·5 million]). From 1990 to 2015, attributable DALYs declined for micronutrient deficiencies, childhood undernutrition, unsafe sanitation and water, and household air pollution; reductions in risk-deleted DALY rates rather than reductions in exposure drove these declines. Rising exposure contributed to notable increases in attributable DALYs from high BMI, high fasting plasma glucose, occupational carcinogens, and drug use. Environmental risks and childhood undernutrition declined steadily with SDI; low physical activity, high BMI, and high fasting plasma glucose increased with SDI. In 119 countries, metabolic risks, such as high BMI and fasting plasma glucose, contributed the most attributable DALYs in 2015. Regionally, smoking still ranked among the leading five risk factors for attributable DALYs in 109 countries; childhood underweight and unsafe sex remained primary drivers of early death and disability in much of sub-Saharan Africa. Interpretation: Declines in some key environmental risks have contributed to declines in critical infectious diseases. Some risks appear to be invariant to SDI. Increasing risks, including high BMI, high fasting plasma glucose, drug use, and some occupational exposures, contribute to rising burden from some conditions, but also provide opportunities for intervention. Some highly preventable risks, such as smoking, remain major causes of attributable DALYs, even as exposure is declining. Public policy makers need to pay attention to the risks that are increasingly major contributors to global burden. Funding: Bill & Melinda Gates Foundation

Generation of murine bone marrow and fetal liver chimeras

The generation of radiation chimeras allows researchers to substitute the hematopoietic system of a mouse with that of one or more donors. A suspension of hematopoietic stem cells (HSCs) is prepared from the bone marrow (BM) or the fetal liver (FL) of a donor mouse and adoptively transferred into an irradiated recipient. Within days, the donor's HSCs will engraft, and their progeny will quickly replace the blood cells of the recipient. This simple tool, together with the large availability of genetically modified mouse lines, can be harnessed to manipulate and study various aspects of blood cell biology in vivo. We present here protocols to generate three types of radiation chimera: (1) BM chimeras, which can assist in determining whether the origin of a genetically based phenotype is the hematopoietic or radio-resistant compartment and which are also conducive for studying the ecology of blood cells and for manipulating the environment hematopoietic cells live; (2) FL chimeras, which allow the study of hematopoietic systems from animals that carry genetic modifications incompatible with postnatal life; and (3) mixed BM chimeras, in which the hematopoietic system comprises blood cells of two different genotypes. Mixed BM chimeras can be used to identify genes that affect hematopoietic cell fitness and to establish whether secreted factors mediate a phenotype of interest. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Generation of bone marrow chimera Basic Protocol 2: Generation of fetal liver chimera Basic Protocol 3: Generation of mixed bone marrow chimera Support Protocol 1: Isolation of bone marrow cells Support Protocol 2: Cell counting by flow cytometry Support Protocol 3: Assessment of chimerism.Stéphane Chappaz, Tahnee L. Saunders, and Benjamin T. Kil

Standardized biomarker and biobanking requirements for personalized psychiatry

With an increasing worldwide phenomenon of psychiatric disorders and treatment regimens with limited efficacy, personalized psychiatry is emerging as a best practice. Within this paradigm, genetic and other biological markers are integrated with clinical data to better define the heterogeneous nature of psychiatric disorders in terms of diagnosis, prognosis, and therapeutic response. Inherent challenges lie not only within the nature of the cohort, but also within the lack of standardization for collection, storage, and analyses of biospecimens and associated clinical data. Biobanks provide global research networks as a valuable resource in the discovery of key molecular signatures underlying mental health disorders. This chapter discusses the significant impact standardization of processes within biobanking and biomarker research will have in supporting advances in personalized psychiatry.Catherine Toben, Victoria K. Arnet, Anita Lo, Pamela H. Saunders, Bernhard T. Baun

The impact of disease on the survival and population growth rate of the Tasmanian devil

1. We investigated the impact of a recently emerged disease, Devil Facial Tumour Disease (DFTD), on the survival and population growth rate of a population of Tasmanian devils, Sarcophilus harrisii, on the Freycinet Peninsula in eastern Tasmania. 2. Cormack-Jolly-Seber and multistate mark-recapture models were employed to investigate the impact of DFTD on age- and sex-specific apparent survival and transition rates. Disease impact on population growth rate was investigated using reverse-time mark-recapture models. 3. The arrival of DFTD triggered an immediate and steady decline in apparent survival rates of adults and subadults, the rate of which was predicted well by the increase in disease prevalence in the population over time. 4. Transitions from healthy to diseased state increased with disease prevalence suggesting that the force of infection in the population is increasing and that the epidemic is not subsiding. 5. The arrival of DFTD coincided with a marked, ongoing decline in the population growth rate of the previously stable population, which to date has not been offset by population compensatory responses

TBK1 and IKKepsilon act redundantly to mediate STING-induced NF-kappaB responses in myeloid cells

Stimulator of Interferon Genes (STING) is a critical component of host innate immune defense but can contribute to chronic autoimmune or autoinflammatory disease. Once activated, the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-STING pathway induces both type I interferon (IFN) expression and nuclear factor-κB (NF-κB)-mediated cytokine production. Currently, these two signaling arms are thought to be mediated by a single upstream kinase, TANK-binding kinase 1 (TBK1). Here, using genetic and pharmacological approaches, we show that TBK1 alone is dispensable for STING-induced NF-κB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IκB kinase ε (IKKε) to drive NF-κB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-κB is significantly less sensitive to TBK1/IKKε kinase inhibition. Our work redefines signaling events downstream of cGAS-STING. Our findings further suggest that cGAS-STING will need to be targeted directly to effectively ameliorate the inflammation underpinning disorders associated with STING hyperactivity.Katherine R. Balka, Cynthia Louis, Tahnee L. Saunders, Amber M. Smith, Dale J. Calleja, Damian B. D’Silva, Fiona Moghaddas, Maximilien Tailler, Kate E. Lawlor, Yifan Zhan, Christopher J. Burns, Ian P. Wicks, Jonathan J. Miner, Benjamin T. Kile, Seth L. Masters, and Dominic De Nard