Monocytes mediate homing of circulating microvesicles to the pulmonary vasculature during low-grade systemic inflammation

Microvesicles (MVs), a plasma membrane-derived subclass of extracellular vesicles, are produced and released into the circulation during systemic inflammation, yet little is known of cell/tissue-specific uptake of MVs under these conditions. We hypothesized that monocytes contribute to uptake of circulating MVs and that their increased margination to the pulmonary circulation and functional priming during systemic inflammation produces substantive changes to the systemic MV homing profile. Cellular uptake of i.v.-injected, fluorescently labelled MVs (J774.1 macrophage-derived) in vivo was quantified by flow cytometry in vascular cell populations of the lungs, liver and spleen of C57BL6 mice. Under normal conditions, both Ly6Chigh and Ly6Clow monocytes contributed to MV uptake but liver Kupffer cells were the dominant target cell population. Following induction of sub-clinical endotoxemia with low-dose i.v. LPS, MV uptake by lung-marginated Ly6Chigh monocytes increased markedly, both at the individual cell level (~2.5-fold) and through substantive expansion of their numbers (~8-fold), whereas uptake by splenic macrophages was unchanged and uptake by Kupffer cells actually decreased (~50%). Further analysis of MV uptake within the pulmonary vasculature using a combined model approach of in vivo macrophage depletion, ex vivo isolated perfused lungs and in vitro lung perfusate cell-based assays, indicated that Ly6Chigh monocytes possess a high MV uptake capacity (equivalent to Kupffer cells), that is enhanced directly by endotoxemia and ablated in the presence of phosphatidylserine (PS)-enriched liposomes and β3 integrin receptor blocking peptide. Accordingly, i.v.-injected PS-enriched liposomes underwent a redistribution of cellular uptake during endotoxemia similar to MVs, with enhanced uptake by Ly6Chigh monocytes and reduced uptake by Kupffer cells. These findings indicate that monocytes, particularly lung-marginated Ly6Chigh subset monocytes, become a dominant target cell population for MVs during systemic inflammation, with significant implications for the function and targeting of endogenous and therapeutically administered MVs, lending novel insights into the pathophysiology of pulmonary vascular inflammation

Efficacy and Safety of Inhaled Carbon Monoxide during Pulmonary Inflammation in Mice

Background: Pulmonary inflammation is a major contributor to morbidity in a variety of respiratory disorders, but treatment options are limited. Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation. Methodology: Mice were exposed to 0–500 ppm inhaled CO for periods of up to 24 hours prior to and following intratracheal instillation of 10 ng LPS. Animals were sacrificed and assessed for intraalveolar neutrophil influx and cytokine levels, flow cytometric determination of neutrophil number and activation in blood, lung and lavage fluid samples, or neutrophil mobilisation from bone marrow. Principal Findings: When administered for 24 hours both before and after LPS, inhaled CO of 100 ppm or more reduced intraalveolar neutrophil infiltration by 40–50%, although doses above 100 ppm were associated with either high carboxyhemoglobin, weight loss or reduced physical activity. This anti-inflammatory effect of CO did not require pre-exposure before induction of injury. 100 ppm CO exposure attenuated neutrophil sequestration within the pulmonary vasculature as well as LPS-induced neutrophilia at 6 hours after LPS, likely due to abrogation of neutrophil mobilisation from bone marrow. In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space

Optimal Tumor Necrosis Factor Induction by Plasmodium falciparum Requires the Highly Localized Release of Parasite Products

Overproduction of tumor necrosis factor (TNF) has been linked with the pathogenesis of Plasmodium falciparum malaria. Here, we examined why the high levels of TNF-inducing activity associated with P. falciparum-parasitized erythrocytes (PE) appear to be lost after cell lysis. Static coculture of PE and peripheral blood mononuclear cells (PBMC), with or without separation by porous membranes, demonstrated that rupture of live PE in the presence of responder cells was required for optimal TNF induction. Although the insoluble fraction of lysed PE was found to partially inhibit TNF responses, supernatants prepared from large numbers of lysed PE still contained only low levels of TNF-inducing activity, which showed no evidence of instability. A dramatic reduction in TNF levels resulted when noncytoadherent PE lines were maintained under low-cell-proximity conditions by suspension coculture. This reduction was much less marked with PE capable of adhering to PBMC, despite the fact that cytoadherent and noncytoadherent parasite lines induced comparable levels of TNF in high-cell-proximity, static coculture. These results suggest that rupture of PE in a highly localized setting, facilitated by either static coculture or the more biologically relevant phenomenon of cytoadherence to PBMC, can result in considerable enhancement of the P. falciparum-induced TNF response

The role of monocytes in sepsis-associated pulmonary microvascular injury

Although monocyte margination to the lungs is enhanced during endotoxemia, little is known of their role in sepsis-associated acute lung injury (ALI). Moreover, monocytes exists as two functionally distinct subsets ‘inflammatory’ Gr-1high and ‘resident’ Gr-1low, which exhibit heterogeneous inflammatory responses upon LPS stimulation both in vivo and in vitro but, the signalling mechanisms regulating their differential responses are unknown. The objective of this research was to define the role of monocyte-endothelial interactions during pulmonary microvascular injury, in response to LPS challenge, using an in vitro monocyte-endothelial coculture model. The specific aims were to: establish a primary lung endothelial cell line culture; investigate priming and enhancement of monocyte pro-inflammatory response; investigate the role of the p38 MAPK pathway in the differential monocyte subset cytokine response, and investigate the role of LPS-mediated TLR4 signalling for monocyte-endothelial reciprocal activation. Primary mouse lung endothelial cells (MLEC), that were successfully cultured, displayed typical morphological and phenotypic properties of the vascular endothelium. During coculture with MLEC, monocytes produced significantly higher levels of TNF when treated with a single high dose of LPS when compared with monocytes cultured alone. Also, upon LPS stimulation, Gr-1high monocytes expressed significantly higher levels of TNF, IL-6, COX-2, and iNOS in comparison to Gr-1low monocytes. Furthermore, significantly higher levels of p38, a critical regulator of inflammatory cytokines, were induced in Gr-1high monocytes in comparison to Gr-1low monocytes. Finally, using a chimeric system of TLR4+/+ (LPS responsive) and TLR4-/- (LPS unresponsive) monocytes and endothelial cells, it was demonstrated that monocytes could activate endothelial cells through TNF-dependent signalling, while endothelial cells could induce TNF production in monocytes. These data indicate an important role for monocytes, especially the Gr-1high subset in the pathogenesis of ALI, and suggest that the p38 MAPK pathway could play a pivotal role in regulating monocyte-induced pulmonary inflammation during sepsis-associated ALI.EThOS - Electronic Theses Online ServiceBiotechnology and Biological Sciences Research Council (BBSRC)GBUnited Kingdo

Investigation of the mechanisms of acute lung injury, using an isolated perfused mouse lung

Acute lung injury (ALI) is a severe inflammatory lung disease with high mortality. Previous studies revealed several important concepts in ALI, including cellular interaction between lung-marginated leukocytes and pulmonary endothelium, and decompartmentalisation of soluble mediators. However, there are inherent limitations within both in vivo and in vitro models to identify the detailed mechanism underlying these concepts. In this PhD project, we attempted to address these unanswered questions, using an in situ isolated perfused mouse lung (IPL). Specifically, we aimed to 1) develop, characterise, and optimise the mouse IPL model; 2) investigate soluble and cellular aspects of two models of ALI that are particularly amenable to study using the IPL, namely ventilator-induced lung injury (VILI) and ischaemia-reperfusion injury. From a physiological viewpoint, VILI consists of 2 primary components, high-stretch and atelectasis. Modelling atelectasis-related injury in vivo is difficult due to negative pleural pressure. We took advantage of the zero pleural pressure of open-chest IPL system to develop an atelectasis-related VILI model. Comparison of this ‘atelectrauma’ and a high-stretch ‘volutrauma’ model demonstrated that both cause lung oedema and pulmonary inflammation, but the inflammatory impact was different between them. Volutrauma, but not atelectrauma, facilitated systemic cytokine release, in which lung-marginated monocytes seem to play an important role. This finding in the VILI model drove us to further investigate the role of these monocytes in an ischaemia-reperfusion model, which is clinically highly relevant and simulates a lung transplantation setting. Our results suggested that lung-marginated monocytes may also contribute to develop ischaemia-reperfusion injury, potentially involving TNF upregulation. Through this PhD project, we have successfully developed a technically very challenging mouse IPL model. We utilised the unique features of the IPL to develop experimental models that we believe will be strong tools to fill the gap between in vivo physiological significance and in vitro mechanistic understanding.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Plasmodium chabaudi chabaudi AS: modification of acute infection in CBA/Ca mice as a result of pre-treatment with erythrocyte band 3 in adjuvant.

In this paper, in vivo data are presented that suggest a role for host recognition of erythrocyte band 3 in the control of malaria parasitaemia. The course of Plasmodium chabaudi chabaudi AS acute infection in CBA/Ca mice was suppressed or enhanced as a result of treatment on two occasions with enriched preparations of normal erythrocyte band 3 in adjuvant. Co-treatment with band 3 and a recombinant polypeptide encoding the C-terminal region of the P. c. chabaudi AS merozoite surface protein 1, which on its own had no clear effect on parasitaemia, appeared to modulate band 3-induced inhibition. Despite several-fold reductions in ascending parasitaemias in some band 3-immunized groups, there was a lack of obvious or unexpected anaemia prior to, or during infection, indicating a degree of specificity in the parasitaemia modifying response for infected rather than uninfected erythrocytes. These findings support a role for modified host recognition of erythrocyte band 3 in the partial immunity that transcends phenotypic and genotypic antigenic variation by malaria parasites

Use of the Alcohol Use Disorders Identification Test (AUDIT) to determine the prevalence of alcohol misuse among HIV-infected individuals.

The aim of the paper is to evaluate alcohol misuse among an inner city adult HIV clinic population with AUDIT (Alcohol Use Disorders Identification Test). A cross-sectional HIV outpatient clinic analysis between 28 February 2011 and 11 March 2011 was carried out. AUDIT, demographic and clinical data were collected. Univariate analysis was performed to look for the associations between variables. Backward stepwise multivariate analyses were performed on significant variables from the univariate analysis to assess for predictors of alcohol dependence. In total, 111 patients were included (60% uptake of clinic attendees); 66% were men and 26% were hepatitis C virus (HCV) co-infected. The median AUDIT score was 5 (within normal range). Thirty-four 'AUDIT positive' cases were identified: five (4.5%) indicated consumption of hazardous levels of alcohol; 21 (19%) indicated harmful levels of alcohol; and eight (7%) were likely alcohol dependent. Younger age (<40 years old) was significantly associated with AUDIT positivity (P = 0.006). On multivariate analysis younger age (P = 0.045, odds ratio 13.8) and lower level of education (P = 0.006, odds ratio 6.7) were predictive of scores indicative of alcohol dependence (AUDIT ≥20). In conclusion, younger age and lower educational levels were associated with scores consistent with alcohol dependence. AUDIT was well tolerated and easy to administer in this outpatient HIV clinic population