Plasmacytoid dendritic cells (pDCs) play a major role in innate immunity producing large amounts of type I interferon following toll-like receptor (TLR) engagement. SOCS2 deficient mice have been shown to be hypersensitive to microbial infection but we found them to be much less susceptible to Sendai viral infection followed by heightened levels of serum type I IFN and reduced viral loads in lungs. To determine the role of SOCS2 in dendritic cell function, we analyzed DCs in peripheral lymphoid organs and tissues both under steady-state and inflammatory conditions. SOCS2-/- mice had markedly elevated pDCs during steady-state and these displayed higher expression of CCR7 and CXCR4. Following Sendai infection, we observed a significantly enhanced recruitment of pDCs into lungs with an elevated CXCR3, and CXCR4 receptor expression and enhanced pulmonary CXCL9, and CXCL12 gene expression on SOCS2 deficient lung tissues. SOCS2 deficiency did not affect pDC development in bone marrow and no overt change was observed in GM-CSF/STATS signalling on bone marrow pDCs. Enhanced ST AT5 early signalling response to GM-CSF stimulation in SOCS2-/- BM-derived and ex vivo pDCs suggests an important role for GM-CSF in promoting pDCs survival in SOCS2-/- animals. In addition, we observed no response of SOCS2-/- bone marrow pDCs and ex vivo pDCs to STAT3 activating cytokine signals (Flt3-L, lL-6). However, an evident level of activated STAT3 protein was observed in bone marrow pDCs and ex vivo pDCs under steady-state conditions. Collectively these results suggest that SOCS2 deficiency may play an important role in modulating viral immunity via promoting pDCs survival under steady-state conditions, and enhanced pDCs recruitment into inflamed organs following Sendai virus infection.

No description available.

GABAergic drugs, such as benzodiazepines, are widely used in clinical practice yet their immune side effects are poorly understood. Preliminary studies have suggested that immune cells express GABAA receptors indicating that they may be controlled by GABA signaling. Herein parallel preclinical, translational and epidemiological approaches are described to help understand the importance of GABAA immunomodulation. The hypothesis is that GABA signaling acts to reduce responsiveness to a pathogen and thus that GABAergic drugs will increase susceptibility to infection. To inform on the clinical importance of this work, data from a subgroup analysis of the Maximizing Efficacy of Targeted Sedation and Reducing Neurological Dysfunction (MENDS) trial (where the relative effects of lorazepam, and dexmedetomidine were compared) are described in septic and non‐septic patients. Consistent with the hypothesis, avoidance of lorazepam sedation decreased mortality by 70% in septic patients but did not affect outcome in non‐septic patients. As preclinical data suggests that benzodiazepines increase mortality at subsedative doses we next conducted a population‐based cohort and nested case‐control design analysis of The Health Improvement Network (THIN), a comprehensive UK general practice database. Benzodiazepines exposure increased the incidence of community acquired pneumonia (CAP) and both 30‐day and long‐term mortality from CAP. Based on these significant accumulating data of the harm of exposure to benzodiazepines during an infection, animal studies were conducted to understand (i) the biological plausibility of our findings and (ii) the mechanism of the effect. In a series of mouse studies the prototypical benzodiazepine, diazepam, increased mortality from Streptococcus pneumoniae through potentiation of GABAA signaling. The increased mortality was associated with increased pathogen load and a delayed cytokine response to the infection. However cellular recruitment was not affected, indicating that local mechanisms were perturbed. Immune cell profiling revealed that alveolar macrophage and monocytes abundantly expressed subunits of the GABAA receptor, compatible with benzodiazepine sensitivity. Ex vivo studies showed that GABAA receptor activation decreased cytokine responses, phagocytosis and bacterial killing by alveolar macrophage likely via inducing an intracellular acidosis. Finally based on the immune cell profile of GABAA receptors we predicted that benzodiazepines that do not target the α1 GABAA subunit would lack the immune suppression observed by nonselective drugs. In accordance with this hypothesis we show that these selective benzodiazepines do not provoke intracellular acidosis, affect cytokine release or bacterial killing of macrophage ex vivo. In vivo the selective benzodiazepine did not increase mortality from infection or increase pathogen load.

Human T-lymphotropic virus Type 1 (HTLV-1) is a retrovirus that persists lifelong in the host. In ~4% of infected people, HTLV-1 causes a chronic disabling neuroinflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is unknown and treatment remains ineffective. In this study we aimed to identify patterns in frequencies of peripheral leukocyte populations and blood gene expression profiles of HTLV-1 carriers that suggest new hypotheses as to the mechanisms of HTLV-1 persistence and HAM/TSP pathology. Flow cytometric immunophenotyping of peripheral blood leukocytes revealed abnormal activation and maturation profiles of effector T cells but not antigen-presenting cells. High frequencies of circulating granzyme and perforin-rich CD8+ T cells were associated with an increased probability of HAM/TSP. However, the cytolytic capacity of these T cells is not known as although they accumulated cytolytic proteins, granzyme mRNA levels were down-regulated in patients with HAM/TSP. Furthermore, presence of HAM/TSP was associated with an expansion of CD56-negative NK cells, which are thought to have decreased cytolytic functions. Blood gene expression profiles identified perturbations of the p53 signalling pathway as a hallmark of HTLV-1 infection. In contrast, a subset of interferon (IFN)-stimulated genes was over-expressed in patients with HAM/TSP but not in asymptomatic HTLV-1 carriers or patients with the clinically similar disease multiple sclerosis. The IFN-inducible signature was present in all circulating leukocytes and its intensity correlated with the clinical severity of HAM/TSP. Leukocytes from patients with HAM/TSP were primed to respond strongly to stimulation with exogenous IFN. However, while type I IFN suppressed expression of the HTLV-1 structural protein Gag it failed to suppress the highly immunogenic viral transcriptional transactivator Tax. Based on our findings we hypothesise that impaired NK cell and T cell-mediated immune responses result in high HTLV-1 proviral loads but that the over-expression of a subset of IFN-stimulated genes contributes to the development of HAM/TSP.

No description available.

The lymphatic system provides a conduit for the trafficking of immune cells from the periphery to draining lymph nodes, both for constitutive immune surveillance and during inflammation. Thus, leucocyte migration into the lymphatics represents an important step in the initiation of a primary immune response, which occurs within lymph nodes. Traditionally, it has been considered that neutrophils are absent from the afferent lymph, having a finite lifespan in the periphery after extravasation from the blood. However, recent research has reported the presence of neutrophils in lymph nodes, in animal models of infection, where neutrophil trafficking was found to occur through afferent lymphatic vessels. This thesis examines the mechanisms regulating neutrophil migration, both by the lymphatic endothelium and neutrophils themselves, under resting and inflammatory conditions. Primary human dermal lymphatic endothelial cells (HDLECs) respond to the pro- inflammatory cytokine, TNF-α by instigating a distinct expression programme, characterised by the up-regulation of various adhesion molecules (ICAM-1, VCAM-1, E-selectin), and CXC- chemokines (ENA-78, GROβ, IL-8), as well as other potential regulators of neutrophil entry such as constitutively expressed adhesion molecules, CD99 and ESAM. Moreover, neutrophils possess counter-receptors for these adhesion molecules and contain basement membrane-specific proteases (elastase) and matrix metalloproteinases (MMPs) such as MMPs -8 and -9, localised in intracellular granules, ready to be exocytosed upon inflammatory stimuli. In vitro data presented in this thesis demonstrate that neutrophil adhesion and transmigration of the lymphatic endothelium is entirely dependent on prior activation of the monolayer with TNF-a. Furthermore, the aforementioned lymphatic-expressed adhesion molecules and chemokines, as well as neutrophil-derived proteases and MMPs are shown to play critical roles in neutrophil adhesion and transmigration of the lymphatic endothelium. Subsequent in vivo experiments confirmed that neutrophil trafficking to lymph nodes across lymphatic vessels is wholly dependent on prior vaccination with Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Tokyo-172. Moreover, neutrophils trafficking to lymph nodes across lymphatic vessels are shown to require ICAM-1. The results described in this thesis provide the first evidence that both the lymphatic endothelium and neutrophils act in concert to regulate entry to lymph nodes and determine the outcome of infection, or vaccination.