Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets
Jessica R. Hitchcock, … , Steve P. Watson, Adam F. Cunningham
Jessica R. Hitchcock, … , Steve P. Watson, Adam F. Cunningham
Published November 16, 2015
Citation Information: J Clin Invest. 2015;125(12):4429-4446. https://doi.org/10.1172/JCI79070.
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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 8

Increased numbers of podoplanin-expressing cells after infection are dependent on IFN-γ and TLR4.

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Increased numbers of podoplanin-expressing cells after infection are dep...
EYFP IFN-γ reporter mice were infected i.p. as above for 7 days, and IFN-γ production was examined by flow cytometry in leukocytes isolated from the liver. (A) Macrophage populations were classified according to the gating strategy outlined in Figure 7. IFN-γ–producing cells were identified by EYFP expression. Representative FACS plots are shown in Supplemental Figure 3. (B) The proportion (at day 7 after infection) of podoplanin+ (left panel) and podoplanin– (right panel) cells in populations 1–8 that are EYFP+. (C) Number of NK cells (CD3– NK1.1+) and IFN-γ+ NK cells per liver in noninfected and day 7–infected mice. (D) Cd1d–/– mice were infected as above, and thrombus development in the liver was examined by H&E at day 7. (E) Tlr4–/– and (F) Ifng–/– mice were infected as above for 7 days, and podoplanin expression (brown) was examined by IHC with F4/80 expression (blue). (G) Leukocytes were isolated from livers of WT and Tlr4–/– mice, and podoplanin expression was measured by flow cytometry. Absolute numbers of podoplanin+ cells from noninfected and day 7–infected mice. All experiments were performed twice with ≥ 4 mice in each group at each time point. Data are shown as mean + SD. Statistical significance was determined relative to noninfected mice (C) or to WT mice (G). *P ≤ 0.05; **P ≤ 0.01, Mann-Whitney sum of ranks test. All images are representative and are from frozen liver sections. Scale bars: 100 μm. Arrows indicate inflammatory lesions.