Bronchiolitis obliterans syndrome is characterized by fibrotic obliteration of small airways which severely impairs graft function and survival after lung transplantation. Bronchial epithelial cells from the transplanted lung can undergo epithelial to mesenchymal transition and this can be accentuated by activated macrophages. Macrophages demonstrate significant plasticity and change phenotype in response to their microenvironment. In this study we aimed to identify secretory products from macrophages that might be therapeutic targets for limiting the inflammatory accentuation of epithelial to mesenchymal transition in bronchiolitis obliterans syndrome. TNFa , IL-1b and IL-8 are elevated in bronchoalveolar lavage from lung transplant patients prior to diagnosis of bronchiolitis obliterans syndrome. Classically activated macrophages secrete more TNFa and IL-1b than alternatively activated macrophages and dramatically accentuate TGF-b 1-driven epithelial to mesenchymal transition in bronchial epithelial cells isolated from lung transplant patients. Blocking TNFa , but not IL-1b , inhibits the accentuation of epithelial to mesenchymal transition. In a pilot unblinded therapeutic intervention in five patients with progressive bronchiolitis obliterans syndrome, anti-TNFa treatment improved forced expiratory volume in 1 second and 6-min walk distances in four patients. Our data identify TNFa as a potential new therapeutic target in bronchiolitis obliterans syndrome deserving of a randomized placebo controlled clinical trial.

Bacterial infections after lung transplantation cause airway epithelial injury and are associated with an increased risk of developing bronchiolitis obliterans syndrome. The damaged epithelium is a source of alarmins that activate the innate immune system, yet their ability to activate fibroblasts in the development of bronchiolitis obliterans syndrome has not been evaluated. Two epithelial alarmins were measured longitudinally in bronchoalveolar lavages from lung transplant recipients who developed bronchiolitis obliterans syndrome and were compared to stable controls. In addition, conditioned media from human airway epithelial cells infected with Pseudomonas aeruginosa was applied to lung fibroblasts and inflammatory responses were determined. Interleukin‐1 alpha (IL‐1α) was increased in bronchoalveolar lavage of lung transplant recipients growing P. aeruginosa (11.5 [5.4–21.8] vs. 2.8 [0.9–9.4] pg/mL, p < 0.01) and was significantly elevated within 3 months of developing bronchiolitis obliterans syndrome (8.3 [1.4–25.1] vs. 3.6 [0.6–17.1] pg/mL, p < 0.01), whereas high mobility group protein B1 remained unchanged. IL‐1α positively correlated with elevated bronchoalveolar lavage IL‐8 levels (r2 = 0.6095, p < 0.0001) and neutrophil percentage (r2 = 0.25, p = 0.01). Conditioned media from P. aeruginosa infected epithelial cells induced a potent pro‐inflammatory phenotype in fibroblasts via an IL‐1α/IL‐1R‐dependent signaling pathway. In conclusion, we propose that IL‐1α may be a novel therapeutic target to limit Pseudomonas associated allograft injury after lung transplantation.

Table 1 Stroke Control Difference (95% CI) p Value Number of participants 27 30 Age (years) Mean 68 58 10 0.001 SD 11 11 4 to 16 Sex Male/female 17/27 15/15 0.13 0.420y Proportion male 0.63 0.50 À0.12 to 0.36 Height (centimetres) Mean 169.6 169.7 À0.1 0.997 SD 7.9 12.2 À5.6 to 5.6 O 2 saturations breathing air (%) Median 97 97 0 0.660* IQR 92 to 98 95 to 98 À1 to 1 Smoking Number ever/ never smoked 13/14 12/18 0.1 0.599y Proportion ever smoked 0.30 0.40 À0.2 to 0.3 Functional residual capacity (litres) Median 2.500 2.780 À0.270 0.003* IQR 2.323 to 3.601 2.258 to 2.898 À0.710 to 0.115 Functional residual capacity (% predicted) Median 76.0 90.0 À14.0 <0.001* IQR 66.5 to 89.5 79.8 to 105.0 À22.0 to À5.0 Peak cough flow rate (litres/min) Mean 297 380 À83 0.019 SD 133 121 À153 to À14 Peak cough flow rate (% predicted PEF) Mean 61.2 86.3 À25.1 <0.001 SD 32.6 17.3 À38.8 to À11.4 Volume inspired before cough (litres) Mean 2.219 3.409 À1.190 <0.001 SD 0.828 0.720 À1.715 to 0.665 Volume Inspired before cough (% predicted VC) Mean 64.3 94.6 À30.1 <0.001 SD 19.5 15.6 À42.2 to 18.5 p Values calculated using t tests except. *p Value calculated using ManneWhitney U test. yp Value calculated using Fisher's exact test. PEF, peak expiratory flow rate; O 2 , oxygen.

manner; no peptide, 2468.56mg/ml, 5 mg, 2181.7626.2 ng/ml (p¼0.006), 10 mg, 15766164.7 ng/ml (p¼0.001), and 20 and 30 mg completely prevented polymer formation in inclusions (p#0.001). Unrelated peptides had no effect. Elastase activity of AT in the supernatant from Z-AT cells was significantly reduced compared to M-AT cells; p#0.001, in keeping with the secretory defect due to retention of Z-AT in inclusion bodies. The elastase activity (and AT concentration) in the supernatant from Z-AT cells was restored by 20 mg 4M, O.D. 405 nm, Z-AT vs Z-AT + 20 mg 4M, 0.12960.009 vs 0.78860.054 respectively, (p#0.001), where a higher O.D. represents higher elastase activity. Functional activity of secreted AT following treatment with 4M was confirmed by its ability to form an SDS-stable complex with elastase as shown by immunoblot. RT-PCR showed that the ER accumulation of Z-AT induced cell stress; NF-kB activation, expression of protein kinase RNA (PKR)-like ER kinase (PERK), and IL-6 (100.4616 pg/ml) and IL-8 (2592.56575 pg/ ml), all of which could be abrogated effectively by 20 mg 4M (IL-6, 45.8628 pg/ml, p#0.001 and IL-8, 184.3629 pg/ml, p¼0.014). These findings are the first evidence that inhibitors of Z-AT polymerisation targeting s4A can prevent its cellular accumulation and deleterious effects. Importantly, this strategy was also able to improve plasma concentration of Z-AT. Acute exacerbations of COPD are the commonest cause of acute medical admissions in the UK and w50% are associated with bacterial infection. Alveolar macrophages (AM) normally clear inhaled bacteria but defective phagocytosis may lead to chronic colonisation and increased exacerbations. Monocyte-derived macrophages (MDM), used to model AM, were obtained from COPD, smoking and healthy subjects. MDM phagocytosis of fluorescently-labelled polystyrene beads, Haemophilus influenzae (HI) or Streptococcus pneumoniae (SP) was measured by fluorimetry. MDM derived from all subjects showed equivalent ability to phagocytose beads, however, COPD and smoker MDM showed significantly reduced phagocytosis of bacteria. Phagocytosis of HI was reduced by 28% and 48% in COPD and smoker MDM respectively, compared to healthy, while SP phagocytosis was reduced by 32% and 52% in COPD and smoker MDM respectively, compared to healthy (Abstract S52 table 1). Having identified defective bacterial phagocytosis in smoker and COPD MDM, the next step was to elucidate the underlying mechanism. Cytoskeletal rearrangement was investigated, with COPD MDM showing significantly reduced phagocytosis of bacteria in comparison to healthy after pre-incubation with nocodazole (microtubule disruptor). Microtubules are involved in membrane trafficking of the phagolysosome and microtubule stability is necessary for effective phagocytosis. Tubulin is acetylated to form stable microtubules and is deacetylated by HDAC6 and Sirt2. COPD MDM showed reduced levels of acetylated tubulin compared to healthy MDM. Pre-incubation with epothilone B (10 nm) a microtubule stabiliser, improved HI phagocytos...

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