Regulation of hepatic inflammation and thrombosis during salmonella infections

Salmonella Typhimurium is one of the most common causes of bacteraemia in children in sub-Saharan Africa and is prevalent in HIV-infected individuals. However, symptoms of this systemic infection are unclear, and while fatalities are frequent, how infection kills is unknown. Here we use a mouse model of systemic (but resolving) infection to investigate physiological and immunological aspects of the host response to infection. The liver is colonised during systemic infection, and in the model used, bacterial numbers peak at day 7 and are largely resolved within a month. Inflammatory lesions, consisting of multiple leukocyte populations, develop within the liver. These persist and are more severe once bacterial clearance is established. Whilst lesions can develop in the absence of T and B cells, these cells contribute to the regulation of inflammatory foci. In the absence of interferon-γ, lesions do not develop and inflammation in the liver is largely absent. In parallel, extensive platelet thrombosis occurs in the liver venous system and the shared kinetics with lesion formation suggest these phenotypes may be co-regulated. Here we describe how parenchymal and vascular inflammation are anchored by inflammatory up-regulation of podoplanin expression in the liver. Thrombosis is substantially abrogated in the absence of C-like lectin-type receptor-2 (CLEC-2) expression on platelets and we show that podoplanin (the physiological ligand for CLEC-2) expression on clodronate-sensitive myeloid populations is necessary for thrombus development. Therefore, the parallel association between inflammation and platelet activation could be the basis for developing novel treatments for systemic bacterial infections in humans

The immunomodulatory effects of the garlic organosulfur compounds allicin and Z-ajoene in an in vitro murine model of LPS-induced inflammation

Cancer is a leading cause of death in the modern world. Chronic inflammation facilitates tumourigenesis and cancer progression by providing an environment conducive to cancer. Dysregulation of the immune response, and particularly inflammation, is an important part of this process. Garlic (Allium sativum) has been used for centuries as both a prophylactic and therapeutic medicinal agent, more recent epidemiological and experimental evidence shows that garlic has both cancer-preventative and immune system-enhancing effects. While garlic contains many bioactive compounds, garlic organosulfur compounds (OSCs) have been most widely studied for their anti-cancer properties. In this study, we hypothesize that garlic OSCs modulate the inflammatory immune response by downregulating pro-inflammatory while stimulating anti-inflammatory responses, preventing the formation of a cancer-friendly chronic inflammatory environment. To test this hypothesis we established and optimised an in vitro inflammatory model using lipopolysaccharide-stimulated RAW264.7 murine macrophages. Expression analysis of selected inflammatory genes was performed by qPCR on RNA harvested 4 h and 8 h post treatment, while protein expression was analysed by ELISA using cell culture supernatant samples harvested 8 h and 24 h post treatment. These experiments were complemented by gene and protein arrays. Results showed that allicin had a more pronounced upregulatory effect on LPS-induced gene expression 4 h post-LPS treatment. In contrast, Z-ajoene generally had mild downregulatory effects on the expression of LPS-induced genes. Conversely, Z-ajoene had pronounced downregulatory effects on LPS-induced inflammatory proteins after 24 h, while allicin showed mild up- or downregulatory effects. Overall, we found that allicin induced an initial pro-inflammatory gene response, while Z-ajoene induced a longer-lasting anti-inflammatory response at a protein level. Finally, as many of the inflammatory genes investigated are regulated by the transcription factor STAT3, we investigated the effects of allicin and Z-ajoene on STAT3 phosphorylation and hence activation. Western blot analyses showed that allicin increased LPS-induced STAT3 phosphorylation (2-8 h), while Z-ajoene was found to decrease the phosphorylation of STAT3 after 4 h. These effects on STAT3 phosphorylation are in agreement with the early pro-inflammatory effect of allicin and the later anti-inflammatory effect of Z-ajoene on LPS-induced gene and protein expression. Further, using Western blotting we showed that E/Z-ajoene directly interacts with and reversibly alkylates STAT3 via a thiol-disulfide reaction with a cysteine thiolate on STAT3

Evaluating the Role of Evapotranspirative Processes for Stormwater Management in Coastal South Carolina Watersheds with Shallow Groundwater

2014 S.C. Water Resources Conference - Informing Strategic Water Planning to Address Natural Resource, Community and Economic Challenge

The immune environment of the mammary gland fluctuates during post-lactational regression and correlates with tumour growth rate

Post-lactational mammary gland regression encompasses extensive programmed cell death and removal of milk-producing epithelial cells, breakdown of extracellular matrix components and redifferentiation of stromal adipocytes. This highly regulated involution process is associated with a transient increased risk of breast cancer in women. Using a syngeneic tumour model, we show that tumour growth is significantly altered depending on the stage of involution at which tumour cells are implanted. Tumour cells injected at day 3 involution grew faster than those in nulliparous mice, whereas tumours initiated at day 6 involution grew significantly slower. These differences in tumour progression correlate with distinct changes in innate immune cells, in particular among F4/80-expressing macrophages and among TCRδ(+) unconventional T cells. Breast cancer post-pregnancy risk is exacerbated in older first-time mothers and, in our model, initial tumour growth is moderately faster in aged mice compared with young mice. Our results have implications for breast cancer risk and the use of anti-inflammatory therapeutics for postpartum breast cancers

Salmonella-induced thrombi in mice develop asynchronously in the spleen and liver and are not effective bacterial traps

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 post-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 to the liver, being evident by 24 h and resolving rapidly thereafter. The distinct kinetics of thrombosis and bacterial burden provide 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

Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods

$\textbf{Background}$: High-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. $\textbf{Methods}$: Multiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3DISCO, SeeDB, CUBIC and PACT. Using confocal, twophoton and light sheet microscopy, their compatibility with wholemount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed. $\textbf{Results}$: Varying 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 wholemount 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. $\textbf{Conclusions}$: The 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 $\textit{in situ}$, and will significantly enhance our understanding of both normal and pathological mammary gland development.This work was supported by a grant from the Medical Research Council (MRC) program grant no. MR/J001023/1 (B.L-L. and C.J.W.). F.M.D. was funded by a National Health and Medical Research Council CJ Martin Biomedical Fellowship (GNT1071074). O.B.H. was funded by a Wellcome Trust PhD Studentship (105377/Z/14/Z). J.R.H was funded by an MRC research grant no. MR/K011014/1. F.C.L. was funded by Cancer Research UK and M.P. was funded by the MRC-LMB (MC_U105178788).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by BioMed Central

Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.

BACKGROUND: High-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. METHODS: Multiple 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. RESULTS: Varying 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. CONCLUSIONS: The 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.This work was supported by a grant from the Medical Research Council (MRC) program grant no. MR/J001023/1 (B.L-L. and C.J.W.). F.M.D. was funded by a National Health and Medical Research Council CJ Martin Biomedical Fellowship (GNT1071074). O.B.H. was funded by a Wellcome Trust PhD Studentship (105377/Z/14/Z). J.R.H was funded by an MRC research grant no. MR/K011014/1. F.C.L. was funded by Cancer Research UK and M.P. was funded by the MRC-LMB (MC_U105178788).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by BioMed Central

Mice Deficient in T-bet Form Inducible NO Synthase-Positive Granulomas That Fail to Constrain Salmonella.

Clearance of intracellular infections caused by Salmonella Typhimurium (STm) requires IFN-γ and the Th1-associated transcription factor T-bet. Nevertheless, whereas IFN-γ-/- mice succumb rapidly to STm infections, T-bet-/- 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 STm-infected IFN-γ-/- and T-bet-/- mice. In IFN-γ-/- 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-γ reverses this, coincident with subsequent granuloma formation and substantially extends survival when compared with mice deficient in all sources of IFN-γ. T-bet-/- mice induce significant levels of IFN-γ- after challenge. Moreover, T-bet-/- 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-/- 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-/- mice, most bacteria are outside these sites. Therefore, Th1 cells act to restrict bacteria within IFN-γ-dependent iNOS+ granulomas and prevent dissemination

Resolving Salmonella infection reveals dynamic and persisting changes in murine bone marrow progenitor cell phenotype and function

The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN-γ-mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN-γ is produced by myeloid, NK, NKT, CD4+ T cells, and some lineage-negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ∼30-fold increase in Sca-1hi progenitors and a corresponding loss of Sca-1lo/int subsets. Most strikingly, the capacity of donor Sca-1hi cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca-1hic-kitint cells have an increased potential to generate B1a-like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life-long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging

Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets

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-ã release enhanced the number of podoplanin-expressing monocytes and Kupffer cells in the hepatic parenchyma and perivascular sites and absence of TLR4, IFN-ã, 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