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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 aeruginosawas 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. aeruginosainfected 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 Pseudomonasassociated allograft injury after lung transplantation.

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. TNFα, IL-1β and IL-8 are elevated in bronchoalveolar lavage from lung transplant patients prior to diagnosis of bronchiolitis obliterans syndrome. Classically activated macrophages secrete more TNFα and IL-1β than alternatively activated macrophages and dramatically accentuate TGF-β1-driven epithelial to mesenchymal transition in bronchial epithelial cells isolated from lung transplant patients. Blocking TNFα, but not IL-1β, inhibits the accentuation of epithelial to mesenchymal transition. In a pilot unblinded therapeutic intervention in five patients with progressive bronchiolitis obliterans syndrome, anti-TNFα treatment improved forced expiratory volume in 1 second and 6-min walk distances in four patients. Our data identify TNFα as a potential new therapeutic target in bronchiolitis obliterans syndrome deserving of a randomized placebo controlled clinical trial.

Epithelial to mesenchymal transition (EMT) has been implicated in the pathogenesis of obliterative bronchiolitis (OB) after lung transplant. Although TNF‐α accentuates TGF‐β1 driven EMT in primary human bronchial epithelial cells (PBECs), we hypothesized that other acute pro‐inflammatory cytokines elevated in the airways of patients with OB may also accentuate EMT and contribute to dysregulated epithelial wound repair. PBECs from lung transplant recipients were stimulated with TGF‐β1 ± IL‐1β, IL‐8, TNF‐α or activated macrophages in co‐culture and EMT assessed. The quality and rate of wound closure in a standardized model of lung epithelial injury was assessed in response to above stimuli. Co‐treatment with TGF‐β1 + TNF‐α or IL‐1β significantly accentuates phenotypic and some functional features of EMT compared to TGF‐β1 alone. Co‐treatment with TGF‐β1 + TNF‐α or IL‐1β accelerates epithelial wound closure however the quality of repair is highly dysregulated. Co‐treatment with TGF‐β1 + IL‐8 has no significant effect on EMT or the speed or quality of wound healing. Activated macrophages dramatically accentuate TGF‐β1‐driven EMT and cause dysregulated wound repair. Crosstalk between macrophage‐derived acute inflammation in the airway and elevated TGF‐β1 may favor dysregulated airway epithelial repair and fibrosis in the lung allograft via EMT.

Chronic lung allograft dysfunction, manifesting as bronchiolitis obliterans syndrome (BOS), is characterized by airway epithelial injury, impaired epithelial regeneration and subsequent airway remodeling. Increased cellular senescence has been reported in renal and liver allografts affected by chronic allograft dysfunction but the significance of cellular senescence in the airway epithelium of the transplanted lung is unknown. Thirty-four lung transplant recipients, 20 with stable graft function and 14 with BOS, underwent transbronchial lung biopsy and histochemical studies for senescence markers in small airways. Compared to nontransplant control lung tissue (n = 9), lung allografts demonstrate significantly increased airway epithelial staining for senescence-associated beta galactosidase (SA β-gal) (p = 0.0215), p16ink4a(p = 0.0002) and p21waf1/cip(p = 0.0138) but there was no difference in expression of these markers between stable and BOS affected recipients (p > 0.05). This preliminary cross-sectional study demonstrates that cellular senescence occurs with increased frequency in the airway epithelium of the lung allograft but does not establish any association between airway epithelial senescence and BOS. A prospective longitudinal study is required to better address any potential causal association between airway epithelial senescence in stable allograft recipients and the subsequent development of BOS.