Helen Maria Marriott

Exposure to respiratory pathogens is a leading cause of exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). Pellino-1 is an E3 ubiquitin ligase known to regulate virally-induced inflammation. We wished to determine the role of Pellino-1 in the host response to respiratory viruses in health and disease. Pellino-1 expression was examined in bronchial sections from patients with GOLD stage 2 COPD and healthy controls. Primary bronchial epithelial cells (PBECs), in which Pellino-1 expression had been knocked down, were extracellularly challenged with the TLR3 agonist poly(I:C). C57BL/6 Peli1-/- mice and wild type littermates were subjected to intranasal infection with clinically-relevant respiratory viruses; rhinovirus (RV1B) and influenza A. We find that Pellino-1 is expressed in the airways of normal subjects and those with COPD, and that Pellino-1 regulates TLR3 signalling and responses to airways viruses. In particular we observed that knockout of Pellino-1 in the murine lung resulted in increased production of proinflammatory cytokines IL-6 and TNFα upon viral infection, accompanied by enhanced recruitment of immune cells to the airways, without any change in viral replication. Pellino-1 therefore regulates inflammatory airway responses without altering replication of respiratory viruses.

Cryptococcus neoformans causes life-threatening infection in the immunocompromised. This and other opportunistic pathogens are an increasing threat as immunosuppression increases globally. To counter antibiotic resistance, there is precedent for developing immune enhancing therapy. However, our understanding of how immunocompetent patients resolve these infections is poor as opportunistic infections typically resolve subclinically. Because this has led to a lack of clinical data, we rely on animal models. Current in vivo infection models either lack mammalian immunity or are not compatible with long term high content imaging required to model the complexities of human host-pathogen interactions. Therefore, we have developed an ex vivo murine precision cut lung slice (PCLS) model to understand innate immunity in cryptococcosis. C57BL/6 mice were sacrificed 0 or 24 hours post infection with KN99α cryptococci. Lungs were inflated with 37°C agarose, 300μm thick PCLS were prepared on a vibratome and imaged by confocal or wide-field fluorescence microscopy. Using PCLS and immunofluorescence, we demonstrate cryptococcal replication and clearance rates are balanced over the first 24 hours of infection. Cell-mediated immunity is alveolar macrophage centric, although alveolar macrophages demonstrate limited phagocytosis of cryptococci and enable intracellular cryptococcal replication. Cryptococcus neoformans responded to the lung environment by forming enlarged cells, although these were not large enough to be titan cells. To further understand cryptococcal proliferation in vivo , we also infected animals with plb1 mutant Cryptococcus neoformans that has been shown to exhibit proliferation defects in vivo . We found no difference in fungal burden with plb1 infected animals 24 hours post infection, but observed significantly larger fungal cells and no incidences of phagocytosis. Thus, the PCLS model can be used to assess the lung immune response early in cryptococcal infection, demonstrating that resident lung macrophages cannot control cryptococcal infection and offer an intracellular niche for Cryptococcus neoformans growth.

Mononuclear phagocytes such as monocytes, tissue-specific macrophages and dendritic cells are primary actors in both innate and adaptive immunity, as well as tissue homoeostasis. They have key roles in a range of physiological and pathological processes, so any strategy targeting these cells will have wide-ranging impact. These phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favouring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis, which is the most pandemic and one of the deadliest disease with one third of the world’s population infected, and 1.8 million deaths worldwide in 2015. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to both circulating monocytes and resident macrophages, using pH sensitive nanoscopic polymersomes made of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-co-poly(2-(di-isopropylamino)ethyl methacrylate) (PMPC-PDPA). Polymersome selectivity to mononuclear phagocytes is demonstrated and ascribed to the polymerised phosphorylcholine motifs affinity toward scavenger receptors. Finally, we demonstrate the successful exploitation of this targeting for the effective eradication of intracellular bacteria that cause tuberculosis Mycobacterium tuberculosis as well as other intracellular parasites including the Mycobacterium bovis, Mycobacterium marinum and the most common bacteria associated with antibiotic resistance, the Staphylococcus aureus .

Background and Purpose: PGE₂inhibits cytokine generation from human lung macrophages. However, the EP receptor that mediates this beneficial anti‐inflammatory effect of PGE₂has not been defined. The aim of this study was to identify the EP receptor by which PGE₂inhibits cytokine generation from human lung macrophages. This was determined by using recently developed EP receptor ligands.Experimental Approach: The effects of PGE₂and EP‐selective agonists on LPS‐induced generation of TNF‐α and IL‐6 from macrophages were evaluated. The effects of EP₂‐selective (PF‐04852946, PF‐04418948) and EP₄‐selective (L‐161,982, CJ‐042794) receptor antagonists on PGE₂responses were studied. The expression of EP receptor subtypes by human lung macrophages was determined by RT‐PCR.Key Results: PGE₂inhibited LPS‐induced andStreptococcus pneumoniae‐induced cytokine generation from human lung macrophages. Analysis of mRNA levels indicated that macrophages expressed EP₂and EP₄receptors. L‐902,688 (EP₄receptor‐selective agonist) was considerably more potent than butaprost (EP₂receptor‐selective agonist) as an inhibitor of TNF‐α generation from macrophages. EP₂receptor‐selective antagonists had marginal effects on the PGE₂inhibition of TNF‐α generation, whereas EP₄receptor‐selective antagonists caused rightward shifts in the PGE₂concentration–response curves.Conclusions and Implications: These studies demonstrate that the EP₄receptor is the principal receptor that mediates the anti‐inflammatory effects of PGE₂on human lung macrophages. This suggests that EP₄receptor agonists could be effective anti‐inflammatory agents in human lung disease.

Macrophages are critical effectors of the early innate response to bacteria in tissues. Phagocytosis and killing of bacteria are interrelated functions essential for bacterial clearance but the rate-limiting step when macrophages are challenged with large numbers of the major medical pathogen Staphylococcus aureus is unknown. We show that macrophages have a finite capacity for intracellular killing and fail to match sustained phagocytosis with sustained microbial killing when exposed to large inocula of S. aureus (Newman, SH1000 and USA300 strains). S. aureus ingestion by macrophages is associated with a rapid decline in bacterial viability immediately after phagocytosis. However, not all bacteria are killed in the phagolysosome, and we demonstrate reduced acidification of the phagolysosome, associated with failure of phagolysosomal maturation and reduced activation of cathepsin D. This results in accumulation of viable intracellular bacteria in macrophages. We show macrophages fail to engage apoptosis-associated bacterial killing. Ultittop mately macrophages with viable bacteria undergo cell lysis, and viable bacteria are released and can be internalized by other macrophages. We show that cycles of lysis and reuptake maintain a pool of viable intracellular bacteria over time when killing is overwhelmed and demonstrate intracellular persistence in alveolar macrophages in the lungs in a murine model.

Lack of reproducibility is an ongoing problem in some areas of the biomedical sciences. Poor experimental design and a failure to engage with experienced statisticians at key stages in the design and analysis of experiments are two factors that contribute to this problem. The RIPOSTE (Reducing IrreProducibility in labOratory STudiEs) framework has been developed to support early and regular discussions between scientists and statisticians in order to improve the design, conduct and analysis of laboratory studies and, therefore, to reduce irreproducibility. This framework is intended for use during the early stages of a research project, when specific questions or hypotheses are proposed. The essential points within the framework are explained and illustrated using three examples (a medical equipment test, a macrophage study and a gene expression study). Sound study design minimises the possibility of bias being introduced into experiments and leads to higher quality research with more reproducible results.

Intracellular killing ofStreptococcus pneumoniaeis complemented by induction of macrophage apoptosis. Here, we show that the toxin pneumolysin (PLY) contributes both to lysosomal/phagolysosomal membrane permeabilization (LMP), an upstream event programing susceptibility to apoptosis, and to apoptosis execution via a mitochondrial pathway, through distinct mechanisms. PLY is necessary but not sufficient for the maximal induction of LMP and apoptosis. PLY’s ability to induce both LMP and apoptosis is independent of its ability to form cytolytic pores and requires only the first three domains of PLY. LMP involves TLR (Toll-like receptor) but not NLRP3/ASC (nucleotide-binding oligomerization domain [Nod]-like receptor family, pyrin domain-containing protein 3/apoptosis-associated speck-like protein containing a caspase recruitment domain) signaling and is part of a PLY-dependent but phagocytosis-independent host response that includes the production of cytokines, including interleukin-1 beta (IL-1β). LMP involves progressive and selective permeability to 40-kDa but not to 250-kDa fluorescein isothiocyanate (FITC)-labeled dextran, as PLY accumulates in the cytoplasm. In contrast, the PLY-dependent execution of apoptosis requires phagocytosis and is part of a host response to intracellular bacteria that also includes NO generation. In cells challenged with PLY-deficient bacteria, reconstitution of LMP using the lysomotrophic detergent LeuLeuOMe favored cell necrosis whereas PLY reconstituted apoptosis. The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP. Following bacterial phagocytosis, PLY triggers apoptosis and prevents macrophage necrosis as a component of a broad-based antimicrobial strategy. This illustrates how a key virulence factor can become the focus of a multilayered and coordinated innate response by macrophages, optimizing pathogen clearance and limiting inflammation.IMPORTANCEStreptococcus pneumoniae, the commonest cause of bacterial pneumonia, expresses the toxin pneumolysin, which can make holes in cell surfaces, causing tissue damage. Macrophages, resident immune cells essential for responses to bacteria in tissues, activate a program of cell suicide called apoptosis, maximizing bacterial clearance and limiting harmful inflammation. We examined pneumolysin’s role in activating this response. We demonstrate that pneumolysin did not directly form holes in cells to trigger apoptosis and show that pneumolysin has two distinct roles which require only part of the molecule. Pneumolysin and other bacterial factors released by bacteria that have not been eaten by macrophages activate macrophages to release inflammatory factors but also make the cell compartment containing ingested bacteria leaky. Once inside the cell, pneumolysin ensures that the bacteria activate macrophage apoptosis, rather than necrosis, enhancing bacterial killing and limiting inflammation. This dual response to pneumolysin is critical for an effective immune response toS. pneumoniae.

Key Points: Neutrophil lifespan is extended in patients with gain-of-function HIF2A mutations. HIF-2α regulates in vivo neutrophil longevity and thus tissue inflammation and repair.