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 11

Thrombi are anchored at sites of endothelial perturbation.

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Thrombi are anchored at sites of endothelial perturbation. WT mice were ...
WT mice were infected i.p. as above for 7 days. (A) Paraffin-embedded liver sections were examined at high magnification by H&E in regions adjacent to the portal vein (left panel) and within the parenchyma (right panel). (B–J) Frozen liver sections were examined by IHC/fluorescent confocal microscopy staining for (B) CD41 (blue), (C) CD31 (green), podoplanin (blue), and Hoechst (gray). Boxed regions are shown at a higher magnification in second, third, and fourth panels. (D) CD11c (brown), CD105 (blue). Left panel shows noninfected, middle panel shows day 7 infected, and right panel shows higher magnification of boxed region. (E) CD31 (green), TF (using antibody 1H1) (red), podoplanin (blue). Top panel shows noninfected, and bottom panel shows day 7 infected. (F) F4/80 (brown), TF (blue; using antibody AF3178). Left panel shows noninfected, and right panel shows day 7 infected; right image is an enlargement of the boxed region. (G) Podoplanin (green), F4/80 (red), and TF (blue; using antibody AF3178) (day 7 infected). (H) WT and Tlr4–/– mice and (I) WT and Pf4-Cre Clec2fl/fl mice were infected as above for 7 days, and liver sections were stained for (H) F4/80 (blue), TF (red; using antibody 1H1), (I) F4/80 (green), TF (red; using antibody 1H1), podoplanin (blue), and (J) fibrin (green), CD41 (red) (WT, day 7 infected). Images in A–G, I, and J are representative of a minimum of 3 experiments, where n ≥ 4 mice per group. Images in H are representative of 2 experiments with ≥ 4 mice per group. Scale bars: 100 μm, unless indicated. Yellow circles indicate areas of compromised endothelial integrity.