Platelet-TLR7 mediates host survival and platelet count during viral infection in the absence of platelet-dependent thrombosis

Abstract

Platelets originate from megakaryocytes in the bone marrow and are released into the circulation as the second-most abundant blood component. Platelets are anucleated, highly granular cell fragments, prepackaged with proteins necessary for their function including small molecules such as serotonin, calcium, and adenosine 5′-diphosphate.1 The cytoplasm of platelets contains precursor-derived messenger RNAs (mRNAs), some of which are translated into proteins.2,3 Canonically, platelets have been associated with hemostasis or initiation of clot formation upon injury of the endothelium, thereby preventing leakage into the interstitial tissue. More recently, platelets have been connected to the host’s immune system via their ability to trap bacteria and present them to leukocytes for destruction.4 Systemic viral infections have long been associated with thrombocytopenia, and single-stranded RNA (ssRNA) viruses such as HIV,5 influenza,6 dengue,7 and hepatitis c virus,8 have been found inside human platelets. Clinical studies have shown that persistence of hepatitis c virus in patients’ platelets, post medical treatment, renders them prone to recurrent infection.8 Platelets are required for clearing infection with lymphocytic choriomeningitis virus and protecting the host against hemorrhagic diathesis.9 Dengue virus, in turn, induces platelet activation through stimulation of apoptotic caspase and mitochondrial dysfunction that explains the observed thrombocytopenia in patients.10 The medical significance of virally induced thrombocytopenia is unclear, as reduction in platelet count may be due to thrombosis or may lead to hemorrhage. In humans, the Toll-like receptor (TLR) family contains 10 known members. TLR2 and TLR4 are surface receptors, whereas TLR3, TLR7, TLR8, and TLR9 are localized to the endosomal compartment of the cell.11 Endosomal TLRs are sensors for nucleic acid ligands: TLR3 is a sensor for double-stranded RNA, TLR9 is a sensor for DNA, and TLR7 is a sensor for ssRNA. Viruses such as encephalomyocarditis virus (EMCV), HIV, and influenza virus, as well as the small guanosine analogs loxoribine (Loxo) and imiquimod, activate the TLR7 receptor.11,12 Influenza virus-induced TLR7 activation requires the acidity of the endosomal compartment, which may facilitate exposure of the viral nucleotides for TLR7 detection. Platelets are known to express functional TLR2,13 TLR4,14 and TLR9,15,16 and activation of TLR2 or TLR4 leads to platelet-neutrophil interaction.13,14 Platelet-leukocyte communication has also been observed during different viral infections.17,,,,-22 Although platelets are known to express TLR2, TLR4, and TLR9, to date, there is no description of functional platelet-TLR7. Given that ssRNA activates TLR7 and that infection with ssRNA viruses is associated with thrombocytopenia, we asked if platelet-TLR7 is actively involved in the decrease of platelet count. We evaluated the presence of TLR7 in platelets and whether its activation or elimination has a functional impact on viral immunity in vivo and/or on thrombotic-platelet responses. Here, we report that human and mouse platelets have a functional TLR7. Further, a 2-hour stimulation of this receptor induces an interaction of platelets with neutrophils and a reduction in platelet count. This process manifests as mild thrombocytopenia, but it is independent of pro-thrombotic behavior. Importantly, during viral infection, platelet presence is necessary to decrease mortality, and TLR7-exppressing platelets are sufficient to increase survival of mice that lack this receptor in all other tissues.