Thrombosis is a common consequence of infection that is associated with poor patient outcome. Nevertheless, the mechanisms by which infection-associated thrombosis is induced, maintained and resolved are poorly understood, as is the contribution thrombosis makes to host control of infection and pathogen spread. The key difference between infection-associated thrombosis and thrombosis in other circumstances is a stronger inflammation-mediated component caused by the presence of the pathogen and its products. This inflammation triggers the activation of platelets, which may accompany damage to the endothelium, resulting in fibrin deposition and thrombus formation. This process is often referred to as thrombo-inflammation. Strikingly, despite its clinical importance and despite thrombi being induced to many different pathogens, it is still unclear whether the mechanisms underlying this process are conserved and how we can best understand this process. This review summarizes thrombosis in a variety of models, including single antigen models such as LPS, and infection models using viruses and bacteria. We provide a specific focus on Salmonella Typhimurium infection as a useful model to address all stages of thrombosis during infection. We highlight how this model has helped us identify how thrombosis can appear in different organs at different times and thrombi be detected for weeks after infection in one site, yet largely be resolved within 24 h in another. Furthermore, we discuss the observation that thrombi induced to Salmonella Typhimurium are largely devoid of bacteria. Finally, we discuss the value of different therapeutic approaches to target thrombosis, the potential importance of timing in their administration and the necessity to maintain normal hemostasis after treatment. Improvements in our understanding of these processes can be used to better target infection-mediated mechanisms of thrombosis.

Thrombosis is a frequent, life-threatening complication of systemic infection associated with multiple organ damage. We have previously described a novel mechanism of inflammation-driven thrombosis induced by Salmonella Typhimurium infection of mice. Thrombosis in the liver develops 7 days after infection, persisting after the infection resolves, and is monocytic cell dependent. Unexpectedly, thrombosis was not prominent in the spleen at this time, despite carrying a similar bacterial burden as the liver. In this study, we show that thrombosis does occur in the spleen but with strikingly accelerated kinetics compared with the liver, being evident by 24 hours and resolving rapidly thereafter. The distinct kinetics of thrombosis and bacterial burden provides a test of the hypothesis that thrombi form in healthy vessels to trap or remove bacteria from the circulation, often termed immunothrombosis. Remarkably, despite bacteria being detected throughout infected spleens and livers in the early days of infection, immunohistological analysis of tissue sections show that thrombi contain very low numbers of bacteria. In contrast, bacteria are present throughout platelet aggregates induced by Salmonella in vitro. Therefore, we show that thrombosis develops with organ-specific kinetics and challenge the universality of immunothrombosis as a mechanism to capture bacteria in vivo.

Systemic immunization with soluble flagellin (sFliC) from Salmonella Typhimurium induces mucosal responses, offering potential as an adjuvant platform for vaccines. Moreover, this engagement of mucosal immunity is necessary for optimal systemic immunity, demonstrating an interaction between these two semi-autonomous immune systems. Although TLR5 and CD103+CD11b+ cDC2 contribute to this process, the relationship between these is unclear in the early activation of CD4+ T cells and the development of antigen-specific B cell responses. In this work, we use TLR5-deficient mice and CD11c-cre.Irf4fl/fl mice (which have reduced numbers of cDC2, particularly intestinal CD103+CD11b+ cDCs), to address these points by studying the responses concurrently in the spleen and the mesenteric lymph nodes (MLN). We show that CD103+CD11b+ cDC2 respond rapidly and accumulate in the MLN after immunization with sFliC in a TLR5-dependent manner. Furthermore, we identify that whilst CD103+CD11b+ cDC2 are essential for the induction of primary T and B cell responses in the mucosa, they do not play such a central role for the induction of these responses in the spleen. Additionally, we show the involvement of CD103+CD11b+ cDC2 in the induction of Th2-associated responses. CD11c-cre.Irf4fl/fl mice showed a reduced primary FliC-specific Th2-associated IgG1 responses, but enhanced Th1-associated IgG2c responses. These data expand our current understanding of the mucosal immune responses promoted by sFliC and highlights the potential of this adjuvant for vaccine usage by taking advantage of the functionality of mucosal CD103+CD11b+ cDC2.

Thrombocytopenia and vascular leakage are clinical hallmarks in dengue hemorrhagic fever. Sung et al. present a new mechanism where platelet-derived extracellular vesicles participate in increasing vascular permeability during dengue virus infection in mice.

Platelets play a key role in the development, progression and resolution of the inflammatory response during sterile inflammation and infection, although the mechanism is not well understood. Here we show that platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from the inflamed tissues. The immune regulatory function of CLEC-2 depends on the expression of its ligand, podoplanin, upregulated on inflammatory macrophages and is independent of platelet activation and secretion. Mechanistically, platelet CLEC-2 and also recombinant CLEC-2-Fc accelerates actin rearrangement and macrophage migration by increasing the expression of podoplanin and CD44, and their interaction with the ERM proteins. During ongoing inflammation, induced by lipopolysaccharide, treatment with rCLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. This is associated with a significant decrease in pro-inflammatory cytokine, TNF-α and an increase in levels of immunosuppressive, IL-10 in the peritoneum. Increased podoplanin expression and actin remodelling favour macrophage migration towards CCL21, a soluble ligand for podoplanin and chemoattractant secreted by lymph node lymphatic endothelial cells. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that platelet CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. These immunomodulatory functions of CLEC-2 are a novel strategy to reduce tissue inflammation and could be therapeutically exploited through rCLEC-2-Fc, to limit the progression to chronic inflammation.

Class I-restricted T cell-associated molecule (CRTAM) is an activation marker expressed on the cell surface of activated invariant natural killer T (iNKT) cells, CD8 T cells, and a small subset of CD4 T cells. CRTAM has also been associated with a proinflammatory profile in murine CD4 T cells. However, CRTAM has not been thoroughly explored in human cells. This work focused on evaluating CRTAM expression in human iNKT lymphocytes after activation with α-galactosylceramide, its widely used specific glycolipid antigen. We also analyzed the involvement of costimulatory molecules in CRTAM expression and whether CRTAM expression is associated with a specific effector cytokine profile. We found that the signal produced by invariant T cell receptor (iTCR) engagement with α-galactosylceramide is sufficient to trigger CRTAM expression on human iNKT cells after 18 h of stimulation. Moreover, we observed a clear association between CRTAM expression and IFN-γ production in iNKT cells from healthy subjects and patients with type 1 diabetes. However, blocking the engagement of costimulatory molecules, such as CD40, CD80, and CD86, did not modify CRTAM expression. These results indicate that CRTAM may also play a role in triggering the production of IFN-γ in human iNKT cells and that CRTAM could be used as a marker to identify these inflammatory cells.

BackgroundClass-I MHC-restricted T cell-associated molecule (CRTAM) is a protein expressed by activated natural killer T (NKT) cells, natural killer (NK) cells, CD8 T cells, and certain CD4 T lymphocytes. It is also expressed in Purkinje neurons and epithelial cells. However, no studies have examined the expression of CRTAM in peripheral blood cells during homeostasis or disease. Therefore, we explored whether CRTAM expression is influenced by the presence of allergic asthma.MethodsWe collected whole peripheral blood cells from non-asthmatic control subjects (n = 17) and patients with asthma (n = 17). All patients with asthma tested positive in allergen skin prick tests. We analyzed CRTAM expression in CD4+ and CD8+ T lymphocyte populations. CRTAM expression was also analyzed in CD177+ neutrophils and IL5Rα+ eosinophils.FindingsThe percentage of CD4+CRTAM+ and CD8+CRTAM+T lymphocytes in peripheral blood was higher in allergic asthma patients compared with healthy controls. Furthermore, the percentage of CD177+CRTAM+ neutrophils in peripheral blood was also elevated in patients with allergic asthma. However, the percentage of IL5Rα+CRTAM+ eosinophils in peripheral blood was not significantly different in patients with allergic asthma compared with healthy controls.ConclusionsCRTAM expression on T cells, eosinophils, and neutrophils may be involved in bronchial inflammation in allergic asthma. Determination of CRTAM expression in peripheral blood may be useful for the diagnosis of bronchial inflammation and/or to identify recently activated immune cells.

Clearance of intracellular infections caused by Salmonella Typhimurium (STm) requires IFN-g and the Th1-associated transcription factor T-bet. Nevertheless, whereas IFN-g 2/2 mice succumb rapidly to STm infections, T-bet 2/2 mice do not. In this study, we assess the anatomy of immune responses and the relationship with bacterial localization in the spleens and livers of STminfected IFN-g 2/2 and T-bet 2/2 mice. In IFN-g 2/2 mice, there is deficient granuloma formation and inducible NO synthase (iNOS) induction, increased dissemination of bacteria throughout the organs, and rapid death. The provision of a source of IFN-g reverses this, coincident with subsequent granuloma formation and substantially extends survival when compared with mice deficient in all sources of IFN-g. T-bet 2/2 mice induce significant levels of IFN-g 2 after challenge. Moreover, T-bet 2/2 mice have augmented IL-17 and neutrophil numbers, and neutralizing IL-17 reduces the neutrophilia but does not affect numbers of bacteria detected. Surprisingly, T-bet 2/2 mice exhibit surprisingly wild-type-like immune cell organization postinfection, including extensive iNOS + granuloma formation. In wild-type mice, most bacteria are within iNOS + granulomas, but in T-bet 2/2 mice, most bacteria are outside these sites. Therefore, Th1 cells act to restrict bacteria within IFN-g-dependent iNOS + granulomas and prevent dissemination.