The identification of markers of macrophage differentiation in PMA-stimulated THP-1 Cells and monocyte-derived macrophages

Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD3) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD3 and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells

How do we evaluate the cost of nosocomial infection? The ECONI protocol: an incidence study with nested case-control evaluating cost and quality of life

Introduction Healthcare-associated or nosocomial infection (HAI) is distressing to patients and costly for the National Health Service (NHS). With increasing pressure to demonstrate cost-effectiveness of interventions to control HAI and notwithstanding the risk from antimicrobial-resistant infections, there is a need to understand the incidence rates of HAI and costs incurred by the health system and for patients themselves. Methods and analysis The Evaluation of Cost of Nosocomial Infection study (ECONI) is an observational incidence survey with record linkage and a nested case-control study that will include postdischarge longitudinal follow-up and qualitative interviews. ECONI will be conducted in one large teaching hospital and one district general hospital in NHS Scotland. The case mix of these hospitals reflects the majority of overnight admissions within Scotland. An incidence survey will record all HAI cases using standard case definitions. Subsequent linkage to routine data sets will provide information on an admission cohort which will be grouped into HAI and non-HAI cases. The case-control study will recruit eligible patients who develop HAI and twice that number without HAI as controls. Patients will be asked to complete five questionnaires: the first during their stay, and four others during the year following discharge from their recruitment admission (1, 3, 6 and 12 months). Multiple data collection methods will include clinical case note review; patient-reported outcome; linkage to electronic health records and qualitative interviews. Outcomes collected encompass infection types; morbidity and mortality; length of stay; quality of life; healthcare utilisation; repeat admissions and postdischarge prescribing. Ethics and dissemination The study has received a favourable ethical opinion from the Scotland A Research Ethics Committee (reference 16/SS/0199). All publications arising from this study will be published in open-access peer-reviewed journal. Lay-person summaries will be published on the ECONI website. Trial registration number NCT03253640; Pre-results

The identification of Staphylococcus aureus factors required for pathogenicity and growth in human blood

Staphylococcus aureus is a human commensal but also has devastating potential as an opportunist pathogen. S. aureus bacteraemia is often associated with an adverse outcome. To identify potential targets for novel control approaches we have identified S. aureus components that are required for growth on human blood. An ordered transposon mutant library was screened, identifying 9 genes involved specifically in haemolysis or growth on human blood agar compared to the parental strain. Three genes (purA, purB and pabA) were subsequently found to be required for pathogenesis in the zebrafish embryo infection model. The pabA growth defect was specific to the red blood cell component of human blood, showing no growth difference compared to the parental strain on human serum, human plasma, sheep or horse blood. PabA is required in the tetrahydrofolate (THF) biosynthesis pathway. The pabA growth defect was found to be due to a combination of loss of THF-dependent dTMP production by the enzyme ThyA and an increased demand for pyrimidines in human blood. Our work highlights pabA and the pyrimidine salvage pathway as potential targets for novel therapeutics and suggests a previously undefined role for a human blood factor in the activity of sulphonamide antibiotics

Regulation of neutrophil senescence by microRNAs

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease

Monocytes regulate the mechanism of T-cell death by inducing Fas-mediated apoptosis during bacterial infection.

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+) T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+) T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+) T-cells in PBMC cultures required 'classical' CD14(+) monocytes, which enhanced T-cell activation. CD3(+) T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+) T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease

The Anti-Apoptotic Effect of Respiratory Syncytial Virus on Human Peripheral Blood Neutrophils is Mediated by a Monocyte Derived Soluble Factor

Respiratory Syncytial Virus (RSV) causes annual epidemics of respiratory disease particularly affecting infants. The associated airway inflammation is characterized by an intense neutrophilia. This neutrophilic inflammation appears to be responsible for much of the pathology and symptoms. Previous work from our group had shown that there are factors within the airways of infants with RSV bronchiolitis that inhibit neutrophil apoptosis. This study was undertaken to determine if RSV can directly affect neutrophil survival

Mutations in succinate dehydrogenase B (SDHB) enhance neutrophil survival independent of HIF-1α expression.

status: publishe

Roles of neutrophils in the regulation of the extent of human inflammation through delivery of IL-1 and clearance of chemokines

This study examined the establishment of neutrophilic inflammation in humans. We tested the hypotheses that neutrophil recruitment was associated with local CXCL8 production and that neutrophils themselves might contribute to the regulation of the size of the inflammatory response. Humans were challenged i.d. with endotoxin. Biopsies of these sites were examined for cytokine production and leukocyte recruitment by qPCR and IHC. Additional in vitro models of inflammation examined the ability of neutrophils to produce and sequester cytokines relevant to neutrophilic inflammation. i.d. challenge with 15 ng of a TLR4-selective endotoxin caused a local inflammatory response, in which 1% of the total biopsy area stained positive for neutrophils at 6 h, correlating with 100-fold up-regulation in local CXCL8 mRNA generation. Neutrophils themselves were the major source of the early cytokine IL-1β. In vitro, neutrophils mediated CXCL8 but not IL-1β clearance (>90% clearance of ≤2 nM CXCL8 over 24 h). CXCL8 clearance was at least partially receptor-dependent and modified by inflammatory context, preserved in models of viral infection but reduced in models of bacterial infection. In conclusion, in a human inflammatory model, neutrophils are rapidly recruited and may regulate the size and outcome of the inflammatory response through the uptake and release of cytokines and chemokines in patterns dependent on the underlying inflammatory stimulus