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.
BackgroundHigh-resolution 3D imaging of intact tissue facilitates cellular and subcellular analyses of complex structures within their native environment. However, difficulties associated with immunolabelling and imaging fluorescent proteins deep within whole organs have restricted their applications to thin sections or processed tissue preparations, precluding comprehensive and rapid 3D visualisation. Several tissue clearing methods have been established to circumvent issues associated with depth of imaging in opaque specimens. The application of these techniques to study the elaborate architecture of the mouse mammary gland has yet to be investigated.MethodsMultiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3D imaging of solvent-cleared organs, see deep brain (seeDB), clear unobstructed brain imaging cocktails (CUBIC) and passive clarity technique. Using confocal, two-photon and light sheet microscopy, their compatibility with whole-mount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed.ResultsVarying degrees of optical transparency, tissue preservation and fluorescent signal conservation were observed between the different clearing methods. SeeDB and CUBIC protocols were considered superior for volumetric fluorescence imaging and whole-mount histochemical staining, respectively. Techniques were compatible with 3D imaging on a variety of platforms, enabling visualisation of mammary ductal and lobulo-alveolar structures at vastly improved depths in cleared tissue.ConclusionsThe utility of whole-organ tissue clearing protocols was assessed in the mouse mammary gland. Most methods utilised affordable and widely available reagents, and were compatible with standard confocal microscopy. These techniques enable high-resolution, 3D imaging and phenotyping of mammary cells and tumours in situ, and will significantly enhance our understanding of both normal and pathological mammary gland development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0754-9) contains supplementary material, which is available to authorized users.
During an inflammatory response, lymphocyte recruitment into tissue must be tightly controlled because dysregulated trafficking contributes to the pathogenesis of chronic disease. Here we show that during inflammation and in response to adiponectin, B cells tonically inhibit T cell trafficking by secreting a peptide (PEPITEM) proteolytically derived from 14.3.3.ζδ protein. PEPITEM binds cadherin-15 on endothelial cells, promoting synthesis and release of sphingosine-1 phosphate, which inhibits trafficking of T cells without affecting recruitment of other leukocytes. Expression of adiponectin receptors on B cells and adiponectin induced PEPITEM secretion wanes with age, implying immune senescence of the pathway. Additionally, these changes are evident in individuals with type-1-diabetes or rheumatoid arthritis, and circulating PEPITEM in patient serum is reduced compared to healthy age matched donors. In both diseases, tonic inhibition of T cell trafficking across inflamed endothelium is lost. Importantly, control of patient T cell trafficking is re-established by exogenous PEPITEM. Moreover, in animal models of peritonitis, hepatic I/R injury, Salmonella infection, Uveitis and Sjögren’s Syndrome, PEPITEM could reduce T cell recruitment into inflamed tissues.
Mucosal immunity is poorly activated after systemic immunization with protein Ags. Nevertheless, induction of mucosal immunity in such a manner would be an attractive and simple way to overcome the intrinsic difficulties in delivering Ag to such sites. Flagellin from Salmonella enterica serovar Typhimurium (FliC) can impact markedly on host immunity, in part via its recognition by TLR5. In this study, we show that systemic immunization with soluble FliC (sFliC) drives distinct immune responses concurrently in the mesenteric lymph nodes (MLN) and the spleen after i.p. and s.c. immunization. In the MLN, but not the spleen, sFliC drives a TLR5-dependent recruitment of CD103+ dendritic cells (DCs), which correlates with a diminution in CD103+ DC numbers in the lamina propria. In the MLN, CD103+ DCs carry Ag and are the major primers of endogenous and transgenic T cell priming. A key consequence of these interactions with CD103+ DCs in the MLN is an increase in local regulatory T cell differentiation. In parallel, systemic sFliC immunization results in a pronounced switching of FliC-specific B cells to IgA in the MLN but not elsewhere. Loss of TLR5 has more impact on MLN than splenic Ab responses, reflected in an ablation of IgA, but not IgG, serum Ab titers. Therefore, systemic sFliC immunization targets CD103+ DCs and drives distinct mucosal T and B cell responses. This offers a potential “Trojan horse” approach to modulate mucosal immunity by systemically immunizing with sFliC.
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