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 (VD(3)) 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 VD(3) 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.
Idiopathic pulmonary fibrosis (IPF) is a complex lung disease characterized by scarring of the lung that is believed to result from an atypical response to injury of the epithelium. Genome-wide ...association studies have reported signals of association implicating multiple pathways including host defense, telomere maintenance, signaling, and cell-cell adhesion.
To improve our understanding of factors that increase IPF susceptibility by identifying previously unreported genetic associations.
We conducted genome-wide analyses across three independent studies and meta-analyzed these results to generate the largest genome-wide association study of IPF to date (2,668 IPF cases and 8,591 controls). We performed replication in two independent studies (1,456 IPF cases and 11,874 controls) and functional analyses (including statistical fine-mapping, investigations into gene expression, and testing for enrichment of IPF susceptibility signals in regulatory regions) to determine putatively causal genes. Polygenic risk scores were used to assess the collective effect of variants not reported as associated with IPF.
We identified and replicated three new genome-wide significant (
< 5 × 10
) signals of association with IPF susceptibility (associated with altered gene expression of
,
, and
) and confirmed associations at 11 previously reported loci. Polygenic risk score analyses showed that the combined effect of many thousands of as yet unreported IPF susceptibility variants contribute to IPF susceptibility.
The observation that decreased
expression associates with increased susceptibility to IPF supports recent studies demonstrating the importance of mTOR signaling in lung fibrosis. New signals of association implicating
and
suggest a possible role of mitotic spindle-assembly genes in IPF susceptibility.
Review of Zebrafish as a tool complementing existing approaches for the study of neutrophil biology, along with the advantages and advances driven by this model.
To understand inflammation and ...immunity, we need to understand the biology of the neutrophil. Whereas these cells can readily be extracted from peripheral blood, their short lifespan makes genetic manipulations impractical. Murine knockout models have been highly informative, and new imaging techniques are allowing neutrophils to be seen during inflammation in vivo for the first time. However, there is a place for a new model of neutrophil biology, which readily permits imaging of individual neutrophils during inflammation in vivo, combined with the ease of genetic and chemical manipulation. The zebrafish has long been the model of choice for the developmental biology community, and the availability of genomic resources and tools for gene manipulation makes this an attractive model. Zebrafish innate immunity shares many features with mammalian systems, including neutrophils with morphological, biochemical, and functional features, also shared with mammalian neutrophils. Transgenic zebrafish with neutrophils specifically labeled with fluorescent proteins have been generated, and this advance has led to the adoption of zebrafish, alongside existing models, by a number of groups around the world. The use of these models has underpinned a number of key advances in the field, including the identification of a tissue gradient of hydrogen peroxide for neutrophil recruitment following tissue injury and direct evidence for reverse migration as a regulatable mechanism of inflammation resolution. In this review, we discuss the importance of zebrafish models in neutrophil biology and describe how the understanding of neutrophil biology has been advanced by the use of these models.
The role of neutrophils in cancer Grecian, Robert; Whyte, Moira K B; Walmsley, Sarah R
British medical bulletin,
12/2018, Letnik:
128, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Abstract
Introduction
It has been known for some time that neutrophils are present in the tumour microenvironment, but only recently have their roles been explored.
Sources of data
Comprehensive ...literature search of neutrophils and cancer (PubMed, Google Scholar and CrossRef) for key articles (systematic reviews, meta-analyses, primary research). References from these articles cross-checked for additional relevant studies.
Areas of agreement
Neutrophils are a heterogeneous population with both pro- and antitumour roles, and display plasticity. Several neutrophil subpopulations have been identified, defined by a combination of features (density, maturity, surface markers, morphology and anatomical site).
Areas of controversy
Limitations in translating murine tumour models to human pathology and paucity of human data. Consensus in defining human neutrophil subpopulations.
Growing points
Neutrophils as therapeutic targets and as possible playmakers in the biological response to newer targeted cancer drugs.
Areas timely for developing research
Understanding the metabolic programming of neutrophils in the tumour microenvironment.
Pulmonary inflammation and bacterial colonization are central to the pathogenesis of chronic obstructive pulmonary disease (COPD). Defects in macrophage phagocytosis of both bacteria and apoptotic ...cells contribute to the COPD phenotype. Small molecule inhibitors with anti-inflammatory activity against p38 mitogen activated protein kinases (MAPKs), phosphatidyl-inositol-3 kinase (PI3K) and Rho kinase (ROCK) are being investigated as novel therapeutics in COPD. Concerns exist, however, about off-target effects. We investigated the effect of p38 MAPK inhibitors (VX745 and SCIO469), specific inhibitors of PI3K α (NVS-P13K-2), δ (NVS-P13K-3) or γ (NVS-P13K-5) and a ROCK inhibitor PF4950834 on macrophage phagocytosis, early intracellular killing of bacteria and efferocytosis of apoptotic neutrophils. Alveolar macrophages (AM) obtained from broncho-alveolar lavage (BAL) or monocyte-derived macrophages (MDM) from COPD patients (GOLD stage II/III) enrolled from a well characterized clinical cohort (MRC COPD-MAP consortium) or from healthy ex-smoker controls were studied. Both COPD AM and MDM exhibited lower levels of bacterial phagocytosis (using Streptococcus pneumoniae and non-typeable Haemophilus influenzae) and efferocytosis than healthy controls. None of the inhibitors altered bacterial internalization or early intracellular bacterial killing in AM or MDM. Conversely PF4950834, but not other inhibitors, enhanced efferocytosis in COPD AM and MDM. These results suggest none of these inhibitors are likely to exacerbate phagocytosis-related defects in COPD, while confirming ROCK inhibitors can enhance efferocytosis in COPD.
The death ligand TRAIL plays a role in physiologic resolution of inflammation and exogenous TRAIL has potential therapeutic benefits in neutrophil‐dominant inflammation.
Novel therapeutics targeting ...neutrophilic inflammation are a major unmet clinical need in acute and chronic inflammation. The timely induction of neutrophil apoptosis is critical for inflammation resolution, and it is thought that acceleration of apoptosis may facilitate resolution at inflammatory sites. We previously demonstrated that a death receptor ligand, TRAIL, accelerates neutrophil apoptosis in vitro. We examined the role of TRAIL in neutrophil‐dominant inflammation in WT and TRAIL‐deficient mice. TRAIL deficiency did not alter constitutive neutrophil apoptosis, whereas exogenous TRAIL accelerated apoptosis of murine peripheral blood neutrophils. We compared TRAIL‐deficient and WT mice in two independent models of neutrophilic inflammation: bacterial LPS‐induced acute lung injury and zymosan‐induced peritonitis. In both models, TRAIL‐deficient mice had an enhanced inflammatory response with increased neutrophil numbers and reduced neutrophil apoptosis. Correction of TRAIL deficiency and supraphysiological TRAIL signaling using exogenous protein enhanced neutrophil apoptosis and reduced neutrophil numbers in both inflammatory models with no evidence of effects on other cell types. These data indicate the potential therapeutic benefit of TRAIL in neutrophilic inflammation.
Cystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear ...whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation.
Blood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR.
CF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF.
CF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.
Hypoxemia is a defining feature of acute respiratory distress syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, yet the resulting tissue hypoxia, and its impact on ...immune responses, is often neglected. In the present study, we have shown that ARDS patients were hypoxemic and monocytopenic within the first 48 h of ventilation. Monocytopenia was also observed in mouse models of hypoxic acute lung injury, in which hypoxemia drove the suppression of type I interferon signaling in the bone marrow. This impaired monopoiesis resulted in reduced accumulation of monocyte-derived macrophages and enhanced neutrophil-mediated inflammation in the lung. Administration of colony-stimulating factor 1 in mice with hypoxic lung injury rescued the monocytopenia, altered the phenotype of circulating monocytes, increased monocyte-derived macrophages in the lung and limited injury. Thus, tissue hypoxia altered the dynamics of the immune response to the detriment of the host and interventions to address the aberrant response offer new therapeutic strategies for ARDS.
Neutrophils are predominantly glycolytic cells that derive little ATP from oxidative phosphorylation; however, they possess an extensive mitochondrial network and maintain a mitochondrial membrane ...potential. Although studies have shown neutrophils need their mitochondria to undergo apoptosis and regulate NETosis, the metabolic role of the respiratory chain in these highly glycolytic cells is still unclear. Recent studies have expanded on the role of reactive oxygen species (ROS) released from the mitochondria as intracellular signaling molecules. Our study shows that neutrophils can use their mitochondria to generate ROS and that mitochondrial ROS release is increased in hypoxic conditions. This is needed for the stabilization of a high level of the critical hypoxic response factor and pro-survival protein HIF-1α in hypoxia. Further, we demonstrate that neutrophils use the glycerol 3-phosphate pathway as a way of directly regulating mitochondrial function through glycolysis, specifically to maintain polarized mitochondria and produce ROS. This illustrates an additional pathway by which neutrophils can regulate HIF-1α stability and will therefore be an important consideration when looking for treatments of inflammatory conditions in which HIF-1α activation and neutrophil persistence at the site of inflammation are linked to disease severity.
The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane ...permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D(-/-) hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function.
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