PB0606 - Stimulation of the ssRNA virus immune receptor, Toll-like receptor 7, on megakaryocytes increases thrombopoesis.

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Amie K. Waller, PhD

Postdoctoral researcher
University of Cambridge
Cambridge, England, United Kingdom

Co-Author(s)

WL

Winnie Lau

Postdoctoral researcher
University of Cambridge
Cambridge, England, United Kingdom
HF

Holly R. Foster

Postdoctoral researcher
Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Jeffrey Cheah Biomedical Centre
Cambridge, England, United Kingdom
AE

Amanda Evans

Research Associate
University of Cambridge
Cambridge, England, United Kingdom
JT

John Tregoning

Reader in Respiratory Infections
Imperial College London
London, England, United Kingdom
AP

Alastair W. Poole

Principal Investigator
School of Physiology, Pharmacology and Neuroscience/University of Bristol
Bristol, England, United Kingdom
AD

Andrew Davidson

Professor of Systems Virology
University of Bristol
Bristol, England, United Kingdom
Cedric Ghevaert, MD PhD

Professor of Transfusion Medicine
Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Jeffrey Cheah Biomedical Centre
Cambridge, England, United Kingdom
JP

James Patterson

Head of Discovery Research
Xap Therapeutics
Cambridge, England, United Kingdom
ML

Moyra Lawrence

Post-Doctoral Fellow
Kyoto University
Kyoto, Kyoto, Japan
AM

Annett Mueller

Research Associate
University of Cambridge
Berlin, Berlin, Germany

Background: TLR7/8 are immune receptors expressed in megakaryocytes which detects single-stranded RNA viruses such as SARS-CoV-2. There is increasing evidence that in addition to raised platelet counts, severe infection with SARS-CoV-2 increases the risk of venous, arterial and microvascular coagulation.

Aims: To determine if ssRNA viruses are capable of increasing thrombopoeisis through direct interaction with megakaryocytes.

Methods: Cells were incubated with and without Gardiquimod (GDQ), a specific agonist of TLR7/8 in cord blood derived (CBMKs) and mouse bone marrow derived megakaryocytes (mMKs). TLR7/8-/- iPSC derived megakaryocytes (iPSC-MKs) were produced using CRISPR Cas9 editing of iPSCs and forward programming using an doxycyclin inducible cassette. GFP labelled SARS-CoV-2 virus was incubated with the TLR7/8-/- iPSC-MKs and wild-type iPSC-MKs.

Results: Incubation with GDQ increased platelet production in CBMKs and mMKs, and increased platelet function. Increased platelet counts were seen in mice treated with GDQ, and mice infected with influenza. Incubation with GDQ induced increased expression of IL1β in the parental iPSC-MKs, however in the TLR7-/- and TLR7/8-/- MKs, no increased expression was observed. There was a significant increase in platelet production from the parental iPSC-MKs in response to incubation with GDQ, which was not seen in the TLR7-/- and TLR7/8-/- MKs. Incubation of the GFP-labelled SARS-CoV-2 virus with wild-type MKs did not lead to a significant increase in fluorescence. Only very low level viral sequences were found in the cells post-incubation demonstrating that penetration within the MKs is unlikely to be of significance. Studies are ongoing to ascertain whether SARS-CoV-2 induces outside in signalling leading to changes in transcription within the MKs (such as elevation of IL1β).

Conclusion(s): TLR7/8 agonists, including ssRNA genome viruses, increase platelet production and functionality from megakaryocytes. SARS-CoV-2 does not appear to penetrate and significantly replicate within MKs, however incubation with megakaryocytes did show elevated expression of IL1β.