Targeting the chemokine network in acute respiratory distress syndrome. Andrew E. Williams*, Ricardo J. José, Paul F. Mercer, Jeremy S. Brown and Rachel C. Chambers. Centre for Inflammation and Tissue Repair, UCL Respiratory, University College London, WC1E 6JF, U.K. Results Introduction 5. CCL2 and CCL7 differentially regulate neutrophil infiltration in response to bacterial infection. Following challenge with S. pneumoniae, neutralisation of either CCL2 (a) or CCL7 (b) had differing effects on neutrophil accumulation. Only neutralisation of CCL7 resulted in a significant decrease in neutrophil numbers following infection (4 h post challenge). Importantly, this reduction in neutrophil number did not affect host defence as bacterial cfu remained comparable (c). 6. CCL2 and CCL7 levels are increased in ARDS patients. In order to translate findings from our in vivo models into the human disease setting, CCL2 (a) and CCL7 (b) were measured in BALF obtained from healthy volunteers, volunteers challenged with LPS (50 ng/ml) or from patients with a clinical diagnosis of ARDS. Both CCL2 and CCL7 were elevated in LPS challenged volunteers and ARDS patients. BAL fluid samples were kindly provided by Prof Danny McAuley (Queen’s University Belfast). 7. Neutrophils differentially express chemokine receptors in the blood and BALF of ARDS patients. Chemokine receptor expression was compared between neutrophils isolated from the blood or BALF of patients with ARDS. Compared to blood, neutrophils isolated from BALF exhibited decreased expression of CXCR1 and increased expression of CCR2. 8. Chemokine synergy between IL-8, CCL2 and CCL7. a b c Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by: ▪ alveolar damage ▪ microvascular leak ▪ neutrophilic inflammation ▪ dysregulated coagulation ▪ respiratory failure ▪ hypoxemia The thrombin receptor PAR-1 provides a link between coagulation and inflammation. However, the role of PAR1 in acute lung injury is less clear. Using a combination of transcriptome analysis, antibody neutralisation studies in animal models, and translational studies on human ARDS BAL fluid, we have established that PAR-1 regulates a novel chemokine axis, which in turn mediates neutrophil recruitment into inflamed lung. 1. PAR1 regulates neutrophil accumulation and alveolar leak. In a mouse model of LPS-induced acute lung inflammation, inhibition of PAR1 with a highly specific antagonist (RWJ58259) reduced neutrophil accumulation in the airspaces (a). In addition, PAR1 antagonism reduced alveolar leak (b), measured as the amount of serum albumin recovered from BALF (3 h post challenge) 2. CCL2 and CCL7 expression is reduced following PAR1 antagonism. Low density mRNA array of inflammatory cytokines and chemokines following PAR1 antagonism (a). Decreased protein expression of the CC-chemokines CCL2 (MCP-1) (b) and CCL7 (MCP-3) (c). Of note, there was no reduction in the classical neutrophilic chemokines CXCL1 or CXCL2 (not shown). 3. CCL2 and CCL7 regulate neutrophil recruitment. Following challenge with LPS, treatment of mice with neutralising anti-CCL2 (a) or ant-CCL7 (b) specific antibodies resulted in a significant reduction in neutrophils recovered from BALF (3 h post challenge) 4. Neutrophils are critical for host defence and alveolar damage. The importance of neutrophils during host defence was assessed following infection with Streptococcus pneumoniae. Neutrophil depletion (a and b) resulted in a reduction in alveolar leak (c), measured as serum albumin recovered from the BALF. Neutrophil numbers also correlated with alveolar leak (d). However, depletion of neutrophils comprised host defence (e), as bacterial numbers increased (24 h post challenge). a b a b a b c a b c d e Blood versus BAL fluid Methods Lung inflammation model. BALB/c mice (6-8 weeks) were challenged with LPS (125 mg/kg) or Streptococcus pneumoniae (5x106 cfu) . 1. Mice were injected i.p with PAR1 antagonist RWJ-58259 (5 mg/kg), 30 min after LPS administration. 2. mice received 10 μg (i.n.) CCL2 (R&D Systems) or CCL7 (Peprotech)-specific neutralising antibodies. BAL fluid and lung tissue was analysed at 3 hours. Chemokine analysis. mRNA levels were analysed using Taqman low density array qPCR chips. Protein levels in supernatants were measured by Luminex. CCL2 and CCL7 were measured by ELISA. Flow cytometry. Neutrophils were isolated from the blood or BALF of ARDS patients, gated as CD16+CD14-HLADR- and were cell surface stained for CXCR1, CXCR2, CCR1, CCR2 and CCR3 (R&D). Neutrophil chemotaxis. Human neutrophils were isolated from blood and purified over a Percoll gradient. ChemoTX plates (NeuroProbe) were used throughout. 10 µg/ml anti-human chemokine antibody (R&D Systems) were used. a b a b anti anti-CCL2/CCL7 10 µg/mouse (i.n) BALF from ARDS patients is highly chemotactic for human neutrophils. Neutralisation of CCL2 (a) or CCL7 (b) inhibited neutrophil chemotaxis, while a combination of anti-IL-8 with anti-CCL2 or anti-CCL7 reduced chemotaxis even further, demonstrating synergy between chemokine families. Summary d e A balance exists between providing host defence but avoiding excessive inflammation. Targeting IL-8, CCL2 and/or CCL7 may be a useful approach for treating neutrophilic inflammation associated with ARDS. Inflammation Host Defence email: andrew.williams@ucl.ac.uk @LungLab @AndrewEWilliams web: http://www.ucl.ac.uk/inflammation-and-tissue-repair/