Regulation of interleukin-8 from macrophages by acute hypoxia and hyperoxia : a role in the pathogenesis of the acute respiratory distress syndrome (ARDS)

The acute respiratory distress syndrome (ARDS) is a catastrophic form of acute lung inflammation. Patients with ARDS require support on the intensive care unit (ICU) and the associated mortality approaches 50%. ARDS represents the severe end of a spectrum of lung injury that evolves over a period of hours or days in a subgroup of patients following a major insult such as multiple trauma, sepsis or aspiration. Professor Haslett's group in Edinburgh have undertaken clinical studies in patients in the very early at-risk period of ARDS, soon after the initiating insult. We have shown that in patients with multiple trauma, raised levels of intrapulmonary interleukin-8 (IL-8), but not other inflammatory cytokines, are associated with subsequent progression to ARDS (n=56, P<0.001). IL-8 is a potent chemoattractant and activator of neutrophils, considered to be the primary injurious cell in ARDS. The high IL-8 levels were detected within a few hours (range 0.75 - 4 hr) of the trauma incident. Immunohistochemical analysis implicated the alveolar macrophage as a potent source of intrapulmonary IL-8. The mechanisms by which IL-8 may be rapidly generated in this clinical setting are unknown.Our clinical observations suggest that events occurring in the immediate aftermath of a trauma incident contribute to the generation of IL-8 in macrophages. I hypothesised that clinically relevant physiological events may include:1) A neuro-endocrine 'stress' response to major trauma. This would result in the rapid intrapulmonary and systemic release of clinically relevant stress mediators including catecholamines and neuropeptides that may stimulate the macrophage to generate IL8.1) A neuro-endocrine 'stress' response to major trauma. This would result in the rapid intrapulmonary and systemic release of clinically relevant stress mediators including catecholamines and neuropeptides that may stimulate the macrophage to generate IL8.2) Acute tissue hypoxia and hyperoxia. By the time of sampling, the trauma victims were likely to have undergone a period of sustained tissue hypoxia secondary to headinjury, atelectasis and lung contusion and subsequent resuscitation with delivery of high flow oxygen. I hypothesised that hypoxia / hyperoxia was as direct multiplestimuli or 'hits' to generate IL-8 in macrophages.I aimed to test these hypotheses in studies of cultured human monocyte-derived macrophages and in a novel animal model of acute lung injury.In human-monocyte derived macrophages, I have shown that the stress mediators adrenalin, substance P and macrophage migration inhibitory factor (MIF) do not increase IL-8 production at an early time-point (2 hr). Compared to normoxic controls, acute hypoxia (PO2 ~ 3.5 KPa) increased IL-8 protein release by 1.8-fold by 2 hours and steady-state IL-8 111RNA expression by 30 mins. The multiple hit of hypoxia / hyperoxia was found to be a more potent stimulus for IL-8 generation than hypoxia or hyperoxia alone.The effects of hypoxia / hyperoxia on IL-8 generation were studied in a rabbit model of acute lung injury. Localised bronchoscopic instillation of HC1 into the left lower lobe of an anaesthetised ventilated rabbit resulted in significantly increased IL-8 mRNA and protein expression, neutrophil infiltration into alveolar airspaces and lung in the directly injured lung but not the contralateral 'indirectly' injured lung. Systemic hypoxaemia was induced by reduction in the inspiratory oxygen fraction. Compared to normoxic controls III (arterial PaC>2 ~ 11 KPa), acute hypoxia (Pa02 ~ 5 KPa) for up to 2 hours increased intrapulmonary IL-8 mRNA but not protein expression in the acid-injured lung. Delivery of 100% oxygen for 2 hours (PaC>2 ~ 60 KPa) following acute hypoxia (a multiple-hit), increased both intrapulmonary IL-8 mRNA and IL-8 protein levels. The increase in IL-8 protein was attenuated if the reoxygenation phase was controlled to return arterial PO2 to normoxic levels (-11 KPa).The mechanisms by which hypoxia may rapidly increase IL-8 mRNA expression in monocyte-derived macrophages was further studied in vitro. The rapidity of the response (30 mins) suggested an increase in gene transcription. Electromobility gel-shift assay revealed that hypoxia increased nuclear levels of the IL-8 promoter-binding transcription factors AP-1 and CEBP-P, but not NF-kB, by 15 min exposure. Hypoxia induced macrophage expression of HIF-la, a critical regulator of hypoxic adaptive responses. However cobalt chloride and desferrioxamine, HIF-la-inducing hypoxia mimics, did not upregulate IL-8, suggesting that IL-8 transcription may be HIF-1 independent. Finally it was demonstrated that in contrast to IL-8, hypoxia inhibited expression of a panel of chemokines and cytokines including MCP-1, MlP-la, MIP-ip and TNF-a. Both the pattern of chemokine expression and transcription factor activation with hypoxia differed from that induced by bacterial lipopolysaccharide (LPS), which potently activated NF-kB and upregulated several inflammatory genes.These data support the hypothesis that acute hypoxia / hyperoxia act as multiple-hits in the generation of macrophage-derived IL-8 in vitro and intrapulmonary IL-8 in vivo, representing a potential mechanism for our observation of elevated alveolar IL-8 levels patients with multiple trauma that progress to ARDS. The observation that hypoxia alone rapidly and selectively increased IL-8 mRNA expression suggests that hypoxia may represent a 'priming' stimulus in macrophages, 'arming' the cell for a subsequent second hit such as hyperoxia. Furthermore, the specific chemokine response to hypoxia differs markedly with that observed with LPS implying potentially distinct adaptive responses to hypoxia and infection in the macrophage

Distinct exosomal mirna profiles from balf and lung tissue of copd and ipf patients

Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are chronic, progressive lung ailments that are characterized by distinct pathologies. Early detection biomarkers and disease mechanisms for these debilitating diseases are lacking. Extracellular vesicles (EVs), including exosomes, are small, lipid-bound vesicles attributed to carry proteins, lipids, and RNA molecules to facilitate cell-to-cell communication under normal and diseased conditions. Exosomal miRNAs have been studied in relation to many diseases. However, there is little to no knowledge regarding the miRNA population of bronchoalveolar lavage fluid (BALF) or the lung-tissue-derived exosomes in COPD and IPF. Here, we determined and compared the miRNA profiles of BALF-and lung-tissue-derived exosomes of healthy non-smokers, smokers, and patients with COPD or IPF in independent cohorts. Results: Exosome characterization using NanoSight particle tracking and TEM demonstrated that the BALF-derived exosomes were ~89.85 nm in size with a yield of ~2.95 × 1010 particles/mL in concentration. Lung-derived exosomes were larger in size (~146.04 nm) with a higher yield of ~2.38 × 1011 particles/mL. NGS results identified three differentially expressed miRNAs in the BALF, while there was one in the lung-derived exosomes from COPD patients as compared to healthy non-smokers. Of these, miR-122-5p was three-or five-fold downregulated among the lung-tissue-derived exosomes of COPD patients as compared to healthy non-smokers and smokers, respectively. Interestingly, there were a large number (55) of differentially expressed miRNAs in the lung-tissue-derived exosomes of IPF patients compared to non-smoking controls. Conclusions: Overall, we identified lung-specific miRNAs associated with chronic lung diseases that can serve as potential biomarkers or therapeutic targets

Distinct Exosomal miRNA Profiles from BALF and Lung Tissue of COPD and IPF Patients

Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are chronic, progressive lung ailments that are characterized by distinct pathologies. Early detection biomarkers and disease mechanisms for these debilitating diseases are lacking. Extracellular vesicles (EVs), including exosomes, are small, lipid-bound vesicles attributed to carry proteins, lipids, and RNA molecules to facilitate cell-to-cell communication under normal and diseased conditions. Exosomal miRNAs have been studied in relation to many diseases. However, there is little to no knowledge regarding the miRNA population of bronchoalveolar lavage fluid (BALF) or the lung-tissue-derived exosomes in COPD and IPF. Here, we determined and compared the miRNA profiles of BALF- and lung-tissue-derived exosomes of healthy non-smokers, smokers, and patients with COPD or IPF in independent cohorts. Results: Exosome characterization using NanoSight particle tracking and TEM demonstrated that the BALF-derived exosomes were ~89.85 nm in size with a yield of ~2.95 × 10(10) particles/mL in concentration. Lung-derived exosomes were larger in size (~146.04 nm) with a higher yield of ~2.38 × 10(11) particles/mL. NGS results identified three differentially expressed miRNAs in the BALF, while there was one in the lung-derived exosomes from COPD patients as compared to healthy non-smokers. Of these, miR-122-5p was three- or five-fold downregulated among the lung-tissue-derived exosomes of COPD patients as compared to healthy non-smokers and smokers, respectively. Interestingly, there were a large number (55) of differentially expressed miRNAs in the lung-tissue-derived exosomes of IPF patients compared to non-smoking controls. Conclusions: Overall, we identified lung-specific miRNAs associated with chronic lung diseases that can serve as potential biomarkers or therapeutic targets

Nalbuphine Tablets for Cough in Patients with Idiopathic Pulmonary Fibrosis

BACKGROUND There are no approved therapies for cough in patients with idiopathic pulmonary fibrosis (IPF). In this small crossover trial we administered nalbuphine extended-release tablets (NAL ER) as a potential cough therapy for such patients. METHODS This randomized, double-blind, placebo-controlled, crossover trial involved two 22-day treatment periods (NAL ER!placebo and placebo!NAL ER) separated by a 2-week washout period. NAL ER was started at a dose of 27 mg once daily and was titrated up to 162 mg twice daily at day 16. The primary end point was percent change from baseline in hourly daytime objective cough frequency as measured by an electronic cough monitor. The daytime period was defined as the patient-reported time of awakening and bedtime. Secondary end points included change in objective 24-hour cough frequency, changes in cough frequency, cough severity, and breathlessness, per patient-reported outcomes. RESULTS A total of 41 patients were randomly assigned and received one or more doses of study medication. There was a 75.1% reduction in daytime objective cough frequency during the NAL ER treatment period versus the placebo treatment period of 22.6%, a 52.5 percentage point placebo-adjusted decrease from baseline (P<0.001) at day 21. There was a 76.1% (95% confidence interval, 83.1 to 69.1) decrease in the 24-hour objective cough frequency with NAL ER, versus a 25.3% (43.9 to 6.7) decrease with placebo, a 50.8 percentage point placebo-adjusted change. Nausea, fatigue, constipation, and dizziness were more common with NAL ER than with placebo. CONCLUSIONS In this short-term crossover trial, NAL ER reduced cough in individuals with IPF. Larger and longer trials are needed to assess the impact on cough versus drug adverse effects. (Funded by Trevi Therapeutics; ClinicalTrials.gov number, NCT04030026.

Functional characterisation of human pulmonary monocyte-like cells in lipopolysaccharide-mediated acute lung inflammation.

BACKGROUND: We have previously reported the presence of novel subpopulations of pulmonary monocyte-like cells (PMLC) in the human lung; resident PMLC (rPMLC, HLA-DR(+)CD14(++)CD16(+)cells) and inducible PMLC (iPMLC, HLA-DR(+)CD14(++)CD16(-) cells). iPMLC are significantly increased in bronchoalveolar lavage (BAL) fluid following inhalation of lipopolysaccharide (LPS). We have carried out the first functional evaluation of PMLC subpopulations in the inflamed lung, following the isolation of these cells, and other lineages, from BAL fluid using novel and complex protocols. METHODS: iPMLC, rPMLC, alveolar macrophages (AM), neutrophils, and regulatory T cells were quantified in BAL fluid of healthy subjects at 9 hours post-LPS inhalation (n = 15). Cell surface antigen expression by iPMLC, rPMLC and AM and the ability of each lineage to proliferate and to undergo phagocytosis were investigated using flow cytometry. Basal cytokine production by iPMLC compared to AM following their isolation from BAL fluid and the responsiveness of both cell types following in vitro treatment with the synthetic corticosteroid dexamethasone were assessed. RESULTS: rPMLC have a significantly increased expression of mature macrophage markers and of the proliferation antigen Ki67, compared to iPMLC. Our cytokine data revealed a pro-inflammatory, corticosteroid-resistant phenotype of iPMLC in this model. CONCLUSIONS: These data emphasise the presence of functionally distinct subpopulations of the monocyte/macrophage lineage in the human lung in experimental acute lung inflammation

Galectin-3 inhibitor GB0139 protects against acute lung injury by inhibiting neutrophil recruitment and activation

Rationale: Galectin-3 (Gal-3) drives fibrosis during chronic lung injury, however, its role in acute lung injury (ALI) remains unknown. Effective pharmacological therapies available for ALI are limited; identifying novel concepts in treatment is essential. GB0139 is a Gal-3 inhibitor currently under clinical investigation for the treatment of idiopathic pulmonary fibrosis. We investigate the role of Gal-3 in ALI and evaluate whether its inhibition with GB0139 offers a protective role. The effect of GB0139 on ALI was explored in vivo and in vitro. Methods: The pharmacokinetic profile of intra-tracheal (i.t.) GB0139 was investigated in C57BL/6 mice to support the daily dosing regimen. GB0139 (1–30 µg) was then assessed following acute i.t. lipopolysaccharide (LPS) and bleomycin administration. Histology, broncho-alveolar lavage fluid (BALf) analysis, and flow cytometric analysis of lung digests and BALf were performed. The impact of GB0139 on cell activation and apoptosis was determined in vitro using neutrophils and THP-1, A549 and Jurkat E6 cell lines. Results: GB0139 decreased inflammation severity via a reduction in neutrophil and macrophage recruitment and neutrophil activation. GB0139 reduced LPS-mediated increases in interleukin (IL)-6, tumor necrosis factor alpha (TNFα) and macrophage inflammatory protein-1-alpha. In vitro, GB0139 inhibited Gal-3-induced neutrophil activation, monocyte IL-8 secretion, T cell apoptosis and the upregulation of pro-inflammatory genes encoding for IL-8, TNFα, IL-6 in alveolar epithelial cells in response to mechanical stretch. Conclusion: These data indicate that Gal-3 adopts a pro-inflammatory role following the early stages of lung injury and supports the development of GB0139, as a potential treatment approach in ALI