Effects of TLR Agonists on the Hypoxia-Regulated Transcription Factor HIF-1α and Dendritic Cell Maturation under Normoxic Conditions

Dendritic cells (DC) are professional antigen presenting cells that represent an important link between innate and adaptive immunity. Danger signals such as toll-like receptor (TLR) agonists induce maturation of DC leading to a T-cell mediated adaptive immune response. In this study, we show that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induce the expression of HIF-1α in human monocyte-derived DC under normoxic conditions. On the functional level, inhibition of HIF-1α using chetomin (CTM), YC-1 and digoxin lead to no consistent effect on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1α stabilization or activity through different mechanisms. Stabilization of HIF-1α protein by hypoxia or CoCl2 did not result in maturation of human DC. In addition, we could show that TLR stimulation resulted in an increase of HIF-1α controlled VEGF secretion. These results show that stimulation of human MoDC with exogenous as well as endogenous TLR agonists induces the expression of HIF-1α in a time-dependent manner. Hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Current evidence suggests that different target genes may be affected by HIF-1α under normoxic conditions with physiological roles that differ from those induced by hypoxia

Secretion of Genome-Free Hepatitis B Virus – Single Strand Blocking Model for Virion Morphogenesis of Para-retrovirus

As a para-retrovirus, hepatitis B virus (HBV) is an enveloped virus with a double-stranded (DS) DNA genome that is replicated by reverse transcription of an RNA intermediate, the pregenomic RNA or pgRNA. HBV assembly begins with the formation of an “immature” nucleocapsid (NC) incorporating pgRNA, which is converted via reverse transcription within the maturing NC to the DS DNA genome. Only the mature, DS DNA-containing NCs are enveloped and secreted as virions whereas immature NCs containing RNA or single-stranded (SS) DNA are not enveloped. The current model for selective virion morphogenesis postulates that accumulation of DS DNA within the NC induces a “maturation signal” that, in turn, triggers its envelopment and secretion. However, we have found, by careful quantification of viral DNA and NCs in HBV virions secreted in vitro and in vivo, that the vast majority of HBV virions (over 90%) contained no DNA at all, indicating that NCs with no genome were enveloped and secreted as empty virions (i.e., enveloped NCs with no DNA). Furthermore, viral mutants bearing mutations precluding any DNA synthesis secreted exclusively empty virions. Thus, viral DNA synthesis is not required for HBV virion morphogenesis. On the other hand, NCs containing RNA or SS DNA were excluded from virion formation. The secretion of DS DNA-containing as well as empty virions on one hand, and the lack of secretion of virions containing single-stranded (SS) DNA or RNA on the other, prompted us to propose an alternative, “Single Strand Blocking” model to explain selective HBV morphogenesis whereby SS nucleic acid within the NC negatively regulates NC envelopment, which is relieved upon second strand DNA synthesis

Estradiol Regulates Expression of Estrogen Receptor ERα46 in Human Macrophages

BACKGROUND:Monocytes and macrophages are key innate immune effector cells that produce cytokines and chemokines upon activation. We and others have shown that 17beta-estradiol (E2) has a direct role in the modulation of monocyte and macrophage immune function. However, relatively little is known about the ability of E2 to regulate isoform expression of estrogen receptors (ERs) in these cells. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we quantify expression of ERalpha and ERbeta in human monocytes and macrophages. We also show for the first time that the N-terminal truncated ERalpha variant, ERalpha46, is expressed in both cell types. Promoter utilization studies reveal that transcription of ERalpha in both cell types occurs from upstream promoters E and F. Treatment with E2 induces ERalpha expression in macrophages but has no effect on ERbeta levels in either cell type. During monocyte-to-macrophage differentiation, ERalpha is upregulated in a time-dependent manner. Previous studies by our group demonstrated that E2 treatment attenuates production of the chemokine CXCL8 in an ER-dependent manner. We now show that ERalpha expression levels parallel the ability of E2 to suppress CXCL8 production. CONCLUSIONS/SIGNIFICANCE:This work demonstrates for the first time that human macrophages predominantly express the truncated ER variant ERalphap46, which is estradiol-inducible. This is mediated through usage of the ERalpha F promoter. Alternative promoter usage may account for tissue and cell type-specific differences in estradiol-induced effects on gene expression. These studies signify the importance of ERalpha expression and regulation in the ability of E2 to modulate innate immune responses

Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses

Data Availability Statement: The individual-level data from these studies is not publicly available to main confidentiality. Data generated by the ISARIC4C consortium is available for collaborative analysis projects through an independent data and materials access committee at isaric4c.net/sample_access. Data and samples from the COVID-Clinical Neuroscience Study are available through collaborative research by application through the NIHR bioresource at https://bioresource.nihr.ac.uk/using-our-bioresource/apply-for-bioresource-data-access/. Brain injury marker and immune mediator data are present in the paper and in the source data file. Source data are provided with this paper.To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1–11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely.National Institute for Health and Care Research (NIHR) (CO-CIN-01) and jointly by NIHR and UK Research and Innovation (CV220-169, MC_PC_19059). B.D.M. is supported by the UKRI/MRC (MR/V03605X/1), the MRC/UKRI (MR/V007181/1), MRC (MR/T028750/1) and Wellcome (ISSF201902/3). C.D. is supported by MRC (MC_PC_19044). We would like to thank the University of Liverpool GCP laboratory facility team for Luminex assistance and the Liverpool University Biobank team for all their help, especially Dr. Victoria Shaw, Lara Lavelle-Langham, and Sue Holden. We would like to acknowledge the Liverpool Experimental Cancer Medicine Centre for providing infrastructure support for this research (Grant Reference: C18616/A25153). We acknowledge the Liverpool Centre for Cell Imaging (CCI) for provision of imaging equipment (Dragonfly confocal microscope) and excellent technical assistance (BBSRC grant number BB/R01390X/1). Tom Solomon is supported by The Pandemic Institute and the NIHR Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool. D.K.M. and E.N. are supported by the NIHR Cambridge Biomedical Centre and by NIHR funding to the NIHR BioResource (RG94028 and RG85445), and by funding from Brain Research UK 201819-20. We thank NIHR BioResource volunteers for their participation, and gratefully acknowledge NIHR BioResource centres, NHS Trusts and staff for their contribution. We thank the National Institute for Health and Care Research, NHS Blood and Transplant, and Health Data Research UK as part of the Digital Innovation Hub Programme. Support for title page creation and format was provided by AuthorArranger, a tool developed at the National Cancer Institute. The authors would like to acknowledge the eDRIS team (Public Health Scotland) for their support in obtaining approvals, the provisioning and linking of data and facilitating access to the National Safe Haven. The views expressed are those of the author(s) and not necessarily those of the UKRI, NHS, the NIHR or the Department of Health and Social Care

Viral Coinfections in Hospitalized Coronavirus Disease 2019 Patients Recruited to the International Severe Acute Respiratory and Emerging Infections Consortium WHO Clinical Characterisation Protocol UK Study

Background: We conducted this study to assess the prevalence of viral coinfection in a well characterized cohort of hospitalized coronavirus disease 2019 (COVID-19) patients and to investigate the impact of coinfection on disease severity. Methods: Multiplex real-time polymerase chain reaction testing for endemic respiratory viruses was performed on upper respiratory tract samples from 1002 patients with COVID-19, aged <1 year to 102 years old, recruited to the International Severe Acute Respiratory and Emerging Infections Consortium WHO Clinical Characterisation Protocol UK study. Comprehensive demographic, clinical, and outcome data were collected prospectively up to 28 days post discharge. Results: A coinfecting virus was detected in 20 (2.0%) participants. Multivariable analysis revealed no significant risk factors for coinfection, although this may be due to rarity of coinfection. Likewise, ordinal logistic regression analysis did not demonstrate a significant association between coinfection and increased disease severity. Conclusions: Viral coinfection was rare among hospitalized COVID-19 patients in the United Kingdom during the first 18 months of the pandemic. With unbiased prospective sampling, we found no evidence of an association between viral coinfection and disease severity. Public health interventions disrupted normal seasonal transmission of respiratory viruses; relaxation of these measures mean it will be important to monitor the prevalence and impact of respiratory viral coinfections going forward

Protein kinase C and cardiac dysfunction: a review

Heart failure (HF) is a physiological state in which cardiac output is insufficient to meet the needs of the body. It is a clinical syndrome characterized by impaired ability of the left ventricle to either fill or eject blood efficiently. HF is a disease of multiple aetiologies leading to progressive cardiac dysfunction and it is the leading cause of deaths in both developed and developing countries. HF is responsible for about 73,000 deaths in the UK each year. In the USA, HF affects 5.8 million people and 550,000 new cases are diagnosed annually. Cardiac remodelling (CD), which plays an important role in pathogenesis of HF, is viewed as stress response to an index event such as myocardial ischaemia or imposition of mechanical load leading to a series of structural and functional changes in the viable myocardium. Protein kinase C (PKC) isozymes are a family of serine/threonine kinases. PKC is a central enzyme in the regulation of growth, hypertrophy, and mediators of signal transduction pathways. In response to circulating hormones, activation of PKC triggers a multitude of intracellular events influencing multiple physiological processes in the heart, including heart rate, contraction, and relaxation. Recent research implicates PKC activation in the pathophysiology of a number of cardiovascular disease states. Few reports are available that examine PKC in normal and diseased human hearts. This review describes the structure, functions, and distribution of PKCs in the healthy and diseased heart with emphasis on the human heart and, also importantly, their regulation in heart failure

The impact of viral mutations on recognition by SARS-CoV-2 specific T cells.

We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.This work is supported by the UK Medical Research Council (MRC); Chinese Academy of Medical Sciences(CAMS) Innovation Fund for Medical Sciences (CIFMS), China; National Institute for Health Research (NIHR)Oxford Biomedical Research Centre, and UK Researchand Innovation (UKRI)/NIHR through the UK Coro-navirus Immunology Consortium (UK-CIC). Sequencing of SARS-CoV-2 samples and collation of data wasundertaken by the COG-UK CONSORTIUM. COG-UK is supported by funding from the Medical ResearchCouncil (MRC) part of UK Research & Innovation (UKRI),the National Institute of Health Research (NIHR),and Genome Research Limited, operating as the Wellcome Sanger Institute. T.I.d.S. is supported by a Well-come Trust Intermediate Clinical Fellowship (110058/Z/15/Z). L.T. is supported by the Wellcome Trust(grant number 205228/Z/16/Z) and by theUniversity of Liverpool Centre for Excellence in Infectious DiseaseResearch (CEIDR). S.D. is funded by an NIHR GlobalResearch Professorship (NIHR300791). L.T. and S.C.M.are also supported by the U.S. Food and Drug Administration Medical Countermeasures Initiative contract75F40120C00085 and the National Institute for Health Research Health Protection Research Unit (HPRU) inEmerging and Zoonotic Infections (NIHR200907) at University of Liverpool inpartnership with Public HealthEngland (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.L.T. is based at the University of Liverpool. M.D.P. is funded by the NIHR Sheffield Biomedical ResearchCentre (BRC – IS-BRC-1215-20017). ISARIC4C is supported by the MRC (grant no MC_PC_19059). J.C.K.is a Wellcome Investigator (WT204969/Z/16/Z) and supported by NIHR Oxford Biomedical Research Centreand CIFMS. The views expressed are those of the authors and not necessarily those of the NIHR or MRC

Fatal COVID-19 outcomes are associated with an antibody response targeting epitopes shared with endemic coronaviruses.

The role of immune responses to previously seen endemic coronavirus epitopes in severe acute respiratory coronavirus 2 (SARS-CoV-2) infection and disease progression has not yet been determined. Here, we show that a key characteristic of fatal coronavirus disease (COVID-19) outcomes is that the immune response to the SARS-CoV-2 spike protein is enriched for antibodies directed against epitopes shared with endemic beta-coronaviruses, and has a lower proportion of antibodies targeting the more protective variable regions of the spike. The magnitude of antibody responses to the SARS-CoV-2 full-length spike protein, its domains and subunits, and the SARS-CoV-2 nucleocapsid also correlated strongly with responses to the endemic beta-coronavirus spike proteins in individuals admitted to intensive care units (ICU) with fatal COVID-19 outcomes, but not in individuals with non-fatal outcomes. This correlation was found to be due to the antibody response directed at the S2 subunit of the SARS-CoV-2 spike protein, which has the highest degree of conservation between the beta-coronavirus spike proteins. Intriguingly, antibody responses to the less cross-reactive SARS-CoV-2 nucleocapsid were not significantly different in individuals who were admitted to ICU with fatal and non-fatal outcomes, suggesting an antibody profile in individuals with fatal outcomes consistent with an original antigenic sin type-response