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 9

Thrombosis is dependent upon CLEC-2 expression on platelets.

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Thrombosis is dependent upon CLEC-2 expression on platelets. WT and Pf4-...
WT and Pf4-Cre Clec2fl/fl mice (lacking CLEC-2 expression on platelets) were infected i.p. as above for 7 days. (A) Thrombosis was examined on paraffin-embedded liver sections using H&E, and (B) vascular occlusion was quantified by point counting of large vessels per tissue section. (C) Absolute number of leukocytes isolated from the liver at day 7. (D) Leukocyte infiltration was examined by IHC: CD11c (brown), F4/80 (blue). Bacterial colonization of (E) liver, (F) blood, and (G) spleen at day 7. WT and Pf4-Cre Clec2fl/fl mice were infected as above for 35 days, and bacterial colonization of (H) the liver and (I) spleen were enumerated. (J) Podoplanin expression (brown) was examined in parenchymal (top panels) and vascular (bottom panels) regions of frozen liver sections by IHC with F4/80 (blue) at day 7 after infection. Leukocytes were isolated from the liver at day 7, and podoplanin expression was measured by flow cytometry. Leukocytes were classified by expression of F4/80 and CD11c, as shown in Supplemental Figure 5. (K) Absolute numbers of podoplanin+ leukocyte populations. (L) Gpvi–/– Pf4-Cre Clec2fl/fl mice (lacking both CLEC-2 expression on platelets and constitutive GPVI expression) and (M) Vav1-iCre+ podoplaninfl/fl mice (lacking podoplanin expression on hematopoietic cells) and relevant littermate controls were infected as above, and thrombus development was examined on frozen liver sections by H&E at day 7 after infection. Data in L and M are each representative of 2 experiments where n ≥ 4 mice per group; remaining data and images are representative of experiments performed at least 4 times using groups containing ≥ 4 mice. Graphs show mean + SD. Statistical significance was determined relative to WT mice. *P ≤ 0.05, Mann-Whitney sum of ranks test. Scale bars: 100 μm. Arrows indicate inflammatory lesions.