Pulmonary Endotoxin is Associated with Reflux, Inflammation, and Adverse Outcomes in Lung Transplant Recipients

Abstract

Purpose Gastroesophageal reflux disease (GERD) and aspiration are risk factors for poor long-term outcomes in lung transplant recipients. However, the mechanisms underlying this relationship are poorly understood. We have previously shown that bile acids and bacterial density in the lung allograft are associated with GERD. As such, we hypothesized that products of the gut microbiome, including pathogen associated molecular patterns (PAMPs), are aspirated into the airways of lung transplant recipients resulting in lung injury. Herein, we measure endotoxin, a bacterial PAMP, in large airway bronchial washes (LABW) of lung transplant recipients and assess the association with GERD, aspiration, inflammation, and allograft survival. Methods Endotoxin was measured using the Limulus amebocyte lysate assay in LABW obtained 2-7 months post-transplant in 61 lung transplant recipients. Proximal and total reflux episodes were measured using 24h pH-impendence testing. Bile acids were measured using mass spectrometry and soluble markers of inflammation were measured with multiplex assay. Results LABW endotoxin was higher in samples that had a positive bacterial culture (p=0.08). Among samples that did not have a positive bacterial culture (n=49), endotoxin concentration was associated with proximal reflux episodes (r=0.312; p=0.02), total reflux episodes (r=0.321; p=0.03), total bile acid concentration (r=0.260; p=0.04), and inflammatory mediators IL-6, IL-8, IL-1ß, and CCL5 (Figure A). Among all 61 lung transplant recipients, those with LABW endotoxin levels in the highest tertile were at increased risk of chronic lung allograft dysfunction (CLAD) [HR: 3.35 (1.43-7.84); p=0.003] (Figure B) and death/retransplant [HR: 4.24 (1.38-13.01); p=0.01]. Conclusion In the absence of overt bacterial infection, endotoxin concentration in LABW was associated with reflux, aspiration, and inflammation. Aspiration of PAMPs may explain why reflux is a risk factor for CLAD. Gastroesophageal reflux disease (GERD) and aspiration are risk factors for poor long-term outcomes in lung transplant recipients. However, the mechanisms underlying this relationship are poorly understood. We have previously shown that bile acids and bacterial density in the lung allograft are associated with GERD. As such, we hypothesized that products of the gut microbiome, including pathogen associated molecular patterns (PAMPs), are aspirated into the airways of lung transplant recipients resulting in lung injury. Herein, we measure endotoxin, a bacterial PAMP, in large airway bronchial washes (LABW) of lung transplant recipients and assess the association with GERD, aspiration, inflammation, and allograft survival. Endotoxin was measured using the Limulus amebocyte lysate assay in LABW obtained 2-7 months post-transplant in 61 lung transplant recipients. Proximal and total reflux episodes were measured using 24h pH-impendence testing. Bile acids were measured using mass spectrometry and soluble markers of inflammation were measured with multiplex assay. LABW endotoxin was higher in samples that had a positive bacterial culture (p=0.08). Among samples that did not have a positive bacterial culture (n=49), endotoxin concentration was associated with proximal reflux episodes (r=0.312; p=0.02), total reflux episodes (r=0.321; p=0.03), total bile acid concentration (r=0.260; p=0.04), and inflammatory mediators IL-6, IL-8, IL-1ß, and CCL5 (Figure A). Among all 61 lung transplant recipients, those with LABW endotoxin levels in the highest tertile were at increased risk of chronic lung allograft dysfunction (CLAD) [HR: 3.35 (1.43-7.84); p=0.003] (Figure B) and death/retransplant [HR: 4.24 (1.38-13.01); p=0.01]. In the absence of overt bacterial infection, endotoxin concentration in LABW was associated with reflux, aspiration, and inflammation. Aspiration of PAMPs may explain why reflux is a risk factor for CLAD.