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 5

TLR4 and IFN-γ mediate inflammation and thrombus development.

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TLR4 and IFN-γ mediate inflammation and thrombus development. WT and Tlr...
WT and Tlr4–/– mice were infected i.p. as above for 7 days. (A) Leukocyte infiltration of the liver was examined by IHC: CD11c (brown), F4/80 (blue). (B) Thrombus development was assessed by H&E staining and (C) was quantified by point counting of large vessels per tissue section. (D) Platelet numbers and (E) mean platelet volume were determined in the blood. (F) WT mice were treated with 20 μg LPS, and thrombus development was examined in the liver after 7 days. WT and Ifng–/– mice were infected as above for 7 days, and (G) leukocyte infiltration of the liver was examined by IHC: CD11c (brown), F4/80 (blue). (H) Thrombus development was assessed by H&E staining and (I) was quantified as above. (J) Platelet numbers and (K) mean platelet volume were determined in the blood. Number of leukocytes isolated from the livers of infected WT and (L) Tlr4–/– or (M) Ifng–/– mice and (N and O) bacterial colonization of the liver were enumerated. (P) WT and Tlr4–/– mice were infected as above for 7 days, and leukocytes isolated from livers were cultured for 48 hours. Secreted IFN-γ was measured in culture supernatants by ELISA. Graphs show mean + SD. Experiments were performed at least twice, with each group containing ≥ 4 mice. Statistical significance was determined relative to WT mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, Mann-Whitney sum of ranks test. ND, not detectable. All images are representative; black arrows indicate inflammatory lesions. Scale bars: 100 μm.