Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets
Jessica R. Hitchcock, Charlotte N. Cook, Saeeda Bobat, Ewan A. Ross, Adriana Flores-Langarica, Kate L. Lowe, Mahmood Khan, C. Coral Dominguez-Medina, Sian Lax, Manuela Carvalho-Gaspar, Stefan Hubscher, G. Ed Rainger, Mark Cobbold, Christopher D. Buckley, Tim J. Mitchell, Andrea Mitchell, Nick D. Jones, N. Van Rooijen, Daniel Kirchhofer, Ian R. Henderson, David H. Adams, Steve P. Watson, Adam F. Cunningham
Jessica R. Hitchcock, Charlotte N. Cook, Saeeda Bobat, Ewan A. Ross, Adriana Flores-Langarica, Kate L. Lowe, Mahmood Khan, C. Coral Dominguez-Medina, Sian Lax, Manuela Carvalho-Gaspar, Stefan Hubscher, G. Ed Rainger, Mark Cobbold, Christopher D. Buckley, Tim J. Mitchell, Andrea Mitchell, Nick D. Jones, N. Van Rooijen, Daniel Kirchhofer, Ian R. Henderson, David H. Adams, Steve P. Watson, Adam F. Cunningham
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Research Article Hematology

Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets

Abstract

Thrombosis is a common, life-threatening consequence of systemic infection; however, the underlying mechanisms that drive the formation of infection-associated thrombi are poorly understood. Here, using a mouse model of systemic Salmonella Typhimurium infection, we determined that inflammation in tissues triggers thrombosis within vessels via ligation of C-type lectin–like receptor-2 (CLEC-2) on platelets by podoplanin exposed to the vasculature following breaching of the vessel wall. During infection, mice developed thrombi that persisted for weeks within the liver. Bacteria triggered but did not maintain this process, as thrombosis peaked at times when bacteremia was absent and bacteria in tissues were reduced by more than 90% from their peak levels. Thrombus development was triggered by an innate, TLR4-dependent inflammatory cascade that was independent of classical glycoprotein VI–mediated (GPVI-mediated) platelet activation. After infection, IFN-γ release enhanced the number of podoplanin-expressing monocytes and Kupffer cells in the hepatic parenchyma and perivascular sites and absence of TLR4, IFN-γ, or depletion of monocytic-lineage cells or CLEC-2 on platelets markedly inhibited the process. Together, our data indicate that infection-driven thrombosis follows local inflammation and upregulation of podoplanin and platelet activation. The identification of this pathway offers potential therapeutic opportunities to control the devastating consequences of infection-driven thrombosis without increasing the risk of bleeding.

Authors

Jessica R. Hitchcock, Charlotte N. Cook, Saeeda Bobat, Ewan A. Ross, Adriana Flores-Langarica, Kate L. Lowe, Mahmood Khan, C. Coral Dominguez-Medina, Sian Lax, Manuela Carvalho-Gaspar, Stefan Hubscher, G. Ed Rainger, Mark Cobbold, Christopher D. Buckley, Tim J. Mitchell, Andrea Mitchell, Nick D. Jones, N. Van Rooijen, Daniel Kirchhofer, Ian R. Henderson, David H. Adams, Steve P. Watson, Adam F. Cunningham

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Figure 2

Extensive thrombosis is a consequence of S. Typhimurium infection.

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Extensive thrombosis is a consequence of S. Typhimurium infection. WT mi...
WT mice were infected i.p. with S. Typhimurium as above. (A) H&E staining of paraffin-embedded sections from noninfected and day 7–infected liver. (B) Tile scan of H&E-stained frozen liver sections from noninfected and day 7–infected mice. (C) Thrombi are observed in the portal vein; 7 day–infected paraffin-embedded section. (D) IHC staining of frozen liver sections at day 7 after infection: CD3 (brown), F4/80 (blue); and CD11c (brown), F4/80 (blue). (E) WT mice were infected i.p. or i.v. as above for 7 days. Thrombosis was examined by H&E, and percentage of vessel occlusion was quantified in large vessels. (F–J) WT mice were infected i.p. as above, and enzymes associated with liver injury were measured in the serum throughout infection. Graphs show mean + SD. Data are representative of a minimum of 3 experiments, where n ≥ 4 mice in each group. Scale bars: 100 μm. V, blood vessel; T, thrombus; PV, portal vein; HA, hepatic artery; BD, bile duct; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase.