A method for the in vivo measurement of zebrafish tissue neutrophil lifespan.

Neutrophil function is thought to be regulated, in large part, by limitation of lifespan by apoptosis. A number of studies suggest that circulating neutrophils have a half-life of approximately 6 hours, although contradictory evidence exists. Measuring tissue neutrophil lifespan, however, is more problematic. It is thought that tissue neutrophils survive longer, perhaps with a half-life in the order of 3-5 days, but this has never been directly measured. Zebrafish are an emerging model organism, with several advantages for the study of vertebrate immunity. In zebrafish, neutrophils constitutively assume tissue locations allowing their direct study in vivo. Using a transgenic approach, neutrophils were labelled with a photoconvertible pigment, Kaede. Photoconversion parameters were optimised and the stability of the Kaede confirmed. Individual neutrophils were photoconverted by scanning a confocal 405 nm laser specifically over each cell and their survival monitored for 48 hours, revealing an in vivo half-life for zebrafish tissue neutrophils of around 120 hours (117.7 hrs, 95% CI 95.67-157.8). Laser energy did not extend neutrophil lifespan, and we conclude that this represents a lower bound for the lifespan of a resting tissue neutrophil in the developing zebrafish larva. This is the first direct measurement of the lifespan of an in vivo tissue neutrophil

Increasing prevalence of asthma diagnosis and symptoms in children is confined to mild symptoms

BACKGROUND: The prevalence of childhood asthma is increasing but few studies have investigated trends in asthma severity. We investigated trends in asthma diagnosis and symptom morbidity between an eight year time period in a paired prevalence study. METHODS: All children in one single school year aged 8-9 years in the city of Sheffield were given a parent respondent questionnaire in 1991 and 1999 based on questions from the International Survey of Asthma and Allergy in Children (ISAAC). Data were obtained regarding the prevalence of asthma and wheeze and current (12 month) prevalences of wheeze attacks, speech limiting wheeze, nocturnal cough and wheeze, and exertional symptoms. RESULTS: The response rates in 1991 and 1999 were 4580/5321 (85.3%) and 5011/6021 (83.2%), respectively. There were significant increases between the two surveys in the prevalence of asthma ever (19.9% v 29.7%, mean difference 11.9%, 95% confidence interval (CI) 10.16 to 13.57, p<0.001), current asthma (10.3% v 13.0%, mean difference 2.7%, 95% CI 1.44 to 4.03, p<0.001), wheeze ever (30.3% v 35.8%, mean difference 5.7%, 95% CI 3.76 to 7.56, p<0.001), wheeze in the previous 12 months (17.0% v 19.4%, mean difference 2.5, 95% CI 0.95 to 4.07, p<0.01), and reporting of medication use (16.9% v 20%, mean difference 3.0%, 95% CI 1.46 to 4.62, p<0.001). There were also significant increases in reported hayfever and eczema diagnoses. CONCLUSIONS: Diagnostic labelling of asthma and lifetime prevalence of wheeze has increased. The current 12 month point prevalence of wheeze has increased but this is confined to occasional symptoms. The increased medication rate may be responsible for the static prevalence of severe asthma symptoms. The significant proportion of children receiving medication but reporting no asthma symptoms identified from our 1999 survey suggests that some children are being inappropriately treated or overtreated

A decoy receptor 3 analogue reduces localised defects in phagocyte function in pneumococcal pneumonia

Background. Therapeutic strategies to modulate the host response to bacterial pneumonia are needed to improve outcomes during community-acquired pneumonia. This study used mice with impaired Fas signalling to examine susceptibility to pneumococcal pneumonia and decoy receptor 3 analogue (DcR3-a) to correct factors associated with increased susceptibility. Methods. Wild-type mice and those with varying degrees of impairment of Fas (lpr) or Fas ligand signalling (gld) were challenged with Streptococcus pneumoniae and microbiological and immunological outcomes measured in the presence or absence of DcR3-a. Results. During established pneumonia, neutrophils became the predominant cell in the airway and gld mice were less able to clear bacteria from the lungs, demonstrating localised impairment of pulmonary neutrophil function in comparison to lpr or wild-type mice. T-cells from gld mice had enhanced activation and reduced apoptosis in comparison to wild-type and lpr mice during established pneumonia. Treatment with DcR3-a reduced T-cell activation and corrected the defect in pulmonary bacterial clearance in gld mice. Conclusions. The results suggest that imbalance in tumour necrosis factor superfamily signalling and excessive T-cell activation can impair bacterial clearance in the lung but that DcR3-a treatment can reduce T-cell activation, restore optimal pulmonary neutrophil function and enhance bacterial clearance during S pneumoniae infection

Pseudomonas expression of an oxygen sensing prolyl hydroxylase homologue regulates neutrophil host responses in vitro and in vivo

Background: Pseudomonas species are adapted to evade innate immune responses and can persist at sites of relative tissue hypoxia, including the mucus-plugged airways of patients with cystic fibrosis and bronchiectasis. The ability of these bacteria to directly sense and respond to changes in local oxygen availability is in part consequent upon expression of the 2-oxoglutarate oxygenase, Pseudomonas prolyl hydroxylase (PPHD), which acts on elongation factor Tu (EF-Tu), and is homologous with the human hypoxia inducible factor (HIF) prolyl hydroxylases. We report that PPHD expression regulates the neutrophil response to acute pseudomonal infection. Methods: In vitro co-culture experiments were performed with human neutrophils and PPHD-deficient and wild-type bacteria and supernatants, with viable neutrophil counts determined by flow cytometry. In vivo consequences of infection with PPHD deficient P. aeruginosa were determined in an acute pneumonia mouse model following intra-tracheal challenge. Results: Supernatants of PPHD-deficient bacterial cultures contained higher concentrations of the phenazine exotoxin pyocyanin and induced greater acceleration of neutrophil apoptosis than wild-type PAO1 supernatants in vitro. In vivo infection with PPHD mutants compared to wild-type PAO1 controls resulted in increased levels of neutrophil apoptosis and impaired control of infection, with higher numbers of P. aeruginosa recovered from the lungs of mice infected with the PPHD-deficient strain. This resulted in an overall increase in mortality in mice infected with the PPHD-deficient strain. Conclusions: Our data show that Pseudomonas expression of its prolyl hydroxylase influences the outcome of host-pathogen interactions in vitro and in vivo, demonstrating the importance of considering how both host and pathogen adaptations to hypoxia together define outcomes of infection. Given that inhibitors for the HIF prolyl hydroxylases are in late stage trials for the treatment of anaemia and that the active sites of PPHD and human HIF prolyl hydroxylases are closely related, the results are of current clinical interest

A Zebrafish Compound Screen Reveals Modulation of Neutrophil Reverse Migration as an Anti-Inflammatory Mechanism

Diseases of failed inflammation resolution are common and largely incurable. Therapeutic induction of inflammation resolution is an attractive strategy to bring about healing without increasing susceptibility to infection. However, therapeutic targeting of inflammation resolution has been hampered by a lack of understanding of the underlying molecular controls. To address this drug development challenge, we developed an in vivo screen for proresolution therapeutics in a transgenic zebrafish model. Inflammation induced by sterile tissue injury was assessed for accelerated resolution in the presence of a library of known compounds. Of the molecules with proresolution activity, tanshinone IIA, derived from a Chinese medicinal herb, potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Tanshinone IIA blocked proinflammatory signals in vivo, and its effects are conserved in human neutrophils, supporting a potential role in treating human inflammation and providing compelling evidence of the translational potential of this screening strategy

Semaphorin 3F signaling actively retains neutrophils at sites of inflammation

Neutrophilic inflammation is central to disease pathogenesis, for example, in chronic obstructive pulmonary disease, yet the mechanisms that retain neutrophils within tissues remain poorly understood. With emerging evidence that axon guidance factors can regulate myeloid recruitment and that neutrophils can regulate expression of a class 3 semaphorin, SEMA3F, we investigated the role of SEMA3F in inflammatory cell retention within inflamed tissues. We observed that neutrophils upregulate SEMA3F in response to proinflammatory mediators and following neutrophil recruitment to the inflamed lung. In both zebrafish tail injury and murine acute lung injury models of neutrophilic inflammation, overexpression of SEMA3F delayed inflammation resolution with slower neutrophil migratory speeds and retention of neutrophils within the tissues. Conversely, constitutive loss of sema3f accelerated egress of neutrophils from the tail injury site in fish, whereas neutrophil-specific deletion of Sema3f in mice resulted in more rapid neutrophil transit through the airways, and significantly reduced time to resolution of the neutrophilic response. Study of filamentous-actin (F-actin) subsequently showed that SEMA3F-mediated retention is associated with F-actin disassembly. In conclusion, SEMA3F signaling actively regulates neutrophil retention within the injured tissues with consequences for neutrophil clearance and inflammation resolution

Alveolar macrophage apoptosis-associated bacterial killing helps prevent murine pneumonia

RATIONALE: Antimicrobial resistance challenges therapy of pneumonia. Enhancing macrophage microbicidal responses would combat this problem but is limited by our understanding of how alveolar macrophages (AM) kill bacteria. OBJECTIVES: To define the role and mechanism of AM apoptosis-associated bacterial killing in the lung. METHODS: We generated a unique CD68.hMcl-1 transgenic mouse with macrophage-specific over-expression of the human anti-apoptotic Mcl-1 protein, a factor upregulated in AM from patients at increased risk of community-acquired pneumonia, to address the requirement for apoptosis-associated killing. MEASUREMENTS AND MAIN RESULTS: Wild-type and transgenic macrophages demonstrated comparable ingestion and initial phagolysosomal killing of bacteria. Continued ingestion (for > 12 h) overwhelmed initial killing and a second late-phase microbicidal response killed viable bacteria in wild-type macrophages, but this response was blunted in CD68.hMcl-1 transgenic macrophages. The late-phase of bacterial killing required both caspase-induced generation of mitochondrial reactive oxygen species (mROS) and nitric oxide (NO), whose peak generation coincided with the late-phase of killing. The CD68.hMcl-1 transgene prevented mROS but not NO generation. Apoptosis-associated killing enhanced pulmonary clearance of Streptococcus pneumoniae and Haemophilus influenzae in wild-type but not CD68.hMcl-1 transgenic mice. Bacterial clearance was enhanced in vivo in CD68.hMcl-1 transgenic mice by reconstitution of apoptosis with BH3 mimetics or clodronate-encapsulated liposomes. Apoptosis-associated killing was not activated during Staphylococcus aureus lung infection. CONCLUSIONS: Mcl-1 upregulation prevents macrophage apoptosis-associated killing and establishes that apoptosis-associated killing is required to allow AM to clear ingested bacteria. Engagement of macrophage apoptosis should be investigated as a novel host-based antimicrobial strategy

NRF2 activation reprogrammes defects in oxidative metabolism to restore macrophage function in COPD

Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration–derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2–related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD

The Role Of Semaphorin3f (sema3f) In Neutrophilic Inflammation

Conference Abstract