Interferons Prime Endothelium for TLR-Mediated Prothrombotic Transformation

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.1186-1186
• Main Authors: Sack, Kelsey D, Eaton, Nathan, Tehrani, Maneli Doroudian, Flaumenhaft, Robert
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-189976

Cytokine storm occurs with respiratory infection and is associated with microvascular thrombosis. However, the cytokines responsible for promoting thrombus formation in the pulmonary microvasculature and the mechanisms by which they act are largely unknown. In its initial response to invading viruses or bacteria, pulmonary epithelium and macrophages release a variety of cytokines into the pulmonary circulation. With progressive invasion, the fragile barrier between alveolus and pulmonary capillaries breaks down and endothelial cells are exposed to pathogen-associated molecular patterns (PAMPs), which stimulate endothelial toll-like receptors (TLRs). To identify the cytokines that stimulate endothelial prothrombotic activity, we screened MIP-1β, MCP-1, IL-10, IL-6, IL-1β, TNFα, IFNα, IFNβ, and IFNγ for their ability to synergize with PAMPs in order to stimulate thrombin generation on endothelium. Dose curves showed that IFNs and TNFα were ~1000-fold more potent than the other cytokines at priming endothelium for thrombin generation induced by poly(I:C), a synthetic PAMP that stimulates TLR3 ( Fig. 1). Anti-tissue factor (TF) antibody completely attenuated thrombin generation induced by either IFNα or IFNγ exposure followed by poly(I:C) stimulation. Evaluation of TF expression and FXa generation showed that although exposure of endothelium to IFNs did not itself enhance TF expression or activity, IFNs substantially augmented TF expression and activity in response to poly(I:C). In addition, IFNα and IFNγ significantly inhibited thrombomodulin expression, even in the absence of poly(I:C). To evaluate whether or not IFNs enhance thrombin generation in endothelium stimulated through TLRs other than TLR3, we exposed endothelium to IFNα or IFNγ, followed by stimulation with different TLR-selective agonists. IFNγ exposure enhanced thrombin generation on endothelium in response to the TLR1/2 agonist PAM3CSK4, but IFNα did not have a significant effect, indicating the specificity of coupling to a particular IFN subtype. In contrast, the stimulation of endothelial thrombin generation by the TLR3 agonists, poly(I:C) and poly(A:U), was more potently enhanced by IFNα compared to IFNγ. Stimulation of thrombin generation by TLR4 agonists, including LPS and CRX-527, was significantly enhanced by IFNγ, but not IFNα. This coupling of IFNα with TLR3 agonists is consistent with the known association of viral infection with IFNα stimulation and TLR3 activation. Coupling of IFNγ