American Gut: an Open Platform for Citizen Science Microbiome Research

McDonald D, Hyde E, Debelius JW, et al. American Gut: an Open Platform for Citizen Science Microbiome Research. mSystems. 2018;3(3):e00031-18

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Schematic of mechanism whereby Fas activation may enhance poly I:C-induced IP-10.

<p>In the absence of Fas engagement, poly I:C, acting through both TLR3 and RIG-I, cause the phosphorylation of JNK and p38 MAPK in a FADD dependant manner. This results in AP-1 activation and translocation of AP-1 to the nucleus, where it acts to repress IP-10 production. Dashed lines indicate that the precise position of FADD in this pathway is unknown <b>(A)</b>. Upon Fas activation, FADD is recruited to Fas, reducing poly I:C-induced JNK and p38 MAPK activation. This reduces the levels of AP-1, thereby alleviating AP-1-mediated repression of the IP-10 promoter, resulting in enhanced IP-10 production. <b>(B)</b></p

Fas activation in THP-1-derived macrophages does not induce caspase 3/7 activation.

<p>THP-1-derived macrophages and Jurkat T cells were treated with 100 ng/ml CH11 and/or 20 μg/ml poly I:C for 24 hrs, or with 5 μM staurosporine as indicated. Both caspase 3/7 activation (<b>A</b>) and cell viability (<b>B</b>) were determined after 24 hrs by fluorescence and trypan blue, respectively. Data shown are representative of three independent experiments, with values shown as Mean ± SEM.</p

Stimulation of macrophages with poly I:C increases expression of Fas and FasL.

<p>THP-1 cells were differentiated with 100 μg/ml PMA for 72 hrs. Cells were treated with increasing concentrations of poly I:C, and Fas and FasL expression detected by qRT-PCR after 8 hrs (<b>A, B</b>) and Western blotting after 48 hrs (<b>C</b>). Human monocyte-derived macrophages from three separate donors were treated with poly I:C (20 μg/ml) and Fas and FasL expression detected by qRT-PCR after 8 hrs (<b>D</b>). Results shown are representative of three separate experiments. Values are shown as Mean ± SEM, (n = 3). ** p<0.01 and *** p<0.001.</p

Fas activation suppresses poly I:C-induced activation of MAP Kinases p38 and JNK.

<p>THP-1 macrophages were treated with CH11 1 hr prior to poly I:C stimulation and Western blotting for phosphorylated p38 MAPK, JNK, IκBα and p42/p44 MAPK was performed. Levels of β-actin were assessed to ensure equal loading of protein. Data shown are representative of three independent experiments.</p

The augmentation of poly-I:C-induced IP-10 production following ligation of Fas is mediated by both TLR3 and RIG-I.

<p>THP-1-derived macrophages were treated with 100 ng/ml CH11 for 1 hr followed by infection with Sendai virus (<b>A,B</b>) or stimulation with poly A:U (<b>C</b>) for a further 8 hrs. Changes in IP-10 production were determined by qRT-PCR (<b>A,C</b>) or ELISA (<b>B</b>). Immortalised wild type and TRIF-^/- BMDMs were stimulated with 10 ng/ml murine agonistic Fas antibody (Jo2) for 1 hr followed by stimulation with 20 μg/ml poly I:C for a further 8 hrs, with changes in cytokine expression were detected by qRT-PCR (<b>D</b>). Data shown are a combination of three independent experiments, with values shown as Mean ± SEM. * p<0.05, ** p<0.01 and *** p<0.001.</p

AP-1 negatively regulates the IP-10 promoter in response to poly I:C, with transfection of FADD augmenting AP-1 luciferase activity.

<p>HEK-293/TLR3 cells were transfected with full length IP-10 luciferase plasmids or IP-10 luciferase plasmids containing point deletions in binding sites for NF-κB (κB1, κB2), proximal ISRE or AP-1. A schematic of these putative binding sites is illustrated <b>(A)</b>. 24 hrs post transfection, cells were stimulated with 20 μg/ml poly I:C for 6 hours with IP-10 luciferase activity expressed as fold-change over TK-renilla activity <b>(B)</b>. Cells were transfected with an AP-1 luciferase reporter plasmid and the indicated doses of FADD plasmid. 24 hrs after transfection cells were stimulated with 20 μg/ml poly I:C or 10 ng/ml TNFα for a further 6 hrs. AP-1 activity were measured and expressed as fold-change over TK-renilla activity <b>(C)</b>. Data shown are a combination of three independent experiments, with values shown as Mean ± SEM.</p

IRAK1 Limits TLR3/4- and IFNAR-Driven IL-27 Production through a STAT1-Dependent Mechanism

IL-27 is a cytokine exerting pleiotropic immunomodulatory effects on a broad spectrum of immune cells. Optimal IL-27 production downstream of TLR3/4 ligand stimulation relies on autocrine type I IFN signaling, defining a first and second phase in IL-27 production. This work shows that IL-1 receptor-associated kinase 1 (IRAK1) limits TLR3/4- and IFNAR-induced IL-27 production. At the mechanistic level, we identified IRAK1 as a novel regulator of STAT1, IRF1, and IRF9. We found hyperactivation of STAT1 together with increased nuclear levels of IRF1 and IRF9 in IRAK1-deficient murine macrophages compared with control cells following stimulation with LPS and poly(I:C). IRAK1-deficient human microglial cells showed higher basal levels of STAT1 and STAT2 compared with control cells. Blocking the kinase activity of TBK1/IKK« in IRAK1 knockdown human microglial cells reduced the high basal levels of STAT1/2, uncovering a TBK1/IKK« kinase–dependent mechanism controlling basal levels of STAT1/2. Stimulating IRAK1 knockdown human microglial cells with IFN-b led to increased IL-27p28 expression compared with control cells. In IRAK1-deficient murine macrophages, increased IL-27 levels were detected by ELISA following IFN-b stimulation compared with control macrophages together with increased nuclear levels of p-STAT1, IRF1, and IRF9. Treatment of wild-type and IRAK1-deficient murine macrophages with fludarabine similarly reduced TLR3/4-induced IL-27 cytokine levels. To our knowledge, this work represents the first report placing IRAK1 in the IFNAR pathway and identifies IRAK1 as an important regulator of STAT1, controlling IL-27 production downstream of TLR3/4 and IFNAR signaling pathways