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

ABSTRACT

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. Aim(s) To determine if ssRNA viruses are capable of increasing thrombopoeisis through direct interaction with megakaryocytes. Method(s) 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. Result(s) 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 IL1beta 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 IL1beta). 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 IL1beta. (Figure Presented).