Residual endotoxin induces primary graft dysfunction through ischemia/reperfusion-primed alveolar macrophages
Mahzad Akbarpour, … , G.R. Scott Budinger, Ankit Bharat
Mahzad Akbarpour, … , G.R. Scott Budinger, Ankit Bharat
Published May 19, 2020
Citation Information: J Clin Invest. 2020;130(8):4456-4469. https://doi.org/10.1172/JCI135838.
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Research Article Immunology

Residual endotoxin induces primary graft dysfunction through ischemia/reperfusion-primed alveolar macrophages

Abstract

Despite the widespread use of antibiotics, bacterial pneumonias in donors strongly predispose to the fatal syndrome of primary graft dysfunction (PGD) following lung transplantation. We report that bacterial endotoxin persists in human donor lungs after pathogen is cleared with antibiotics and is associated with neutrophil infiltration and PGD. In mouse models, depletion of tissue-resident alveolar macrophages (TRAMs) attenuated neutrophil recruitment in response to endotoxin as shown by compartmental staining and intravital imaging. Bone marrow chimeric mice revealed that neutrophils were recruited by TRAM through activation of TLR4 in a MyD88-dependent manner. Intriguingly, low levels of endotoxin, insufficient to cause donor lung injury, promoted TRAM-dependent production of CXCL2, increased neutrophil recruitment, and led to PGD, which was independent of donor NCMs. Reactive oxygen species (ROS) increased in human donor lungs starting from the warm-ischemia phase and were associated with increased transcription and translocation to the plasma membrane of TLR4 in donor TRAMs. Consistently, scavenging ROS or inhibiting their production to prevent TLR4 transcription/translocation or blockade of TLR4 or coreceptor CD14 on donor TRAMs prevented neutrophil recruitment in response to endotoxin and ameliorated PGD. Our studies demonstrate that residual endotoxin after successful treatment of donor bacterial pneumonia promotes PGD through ischemia/reperfusion-primed donor TRAMs.

Authors

Mahzad Akbarpour, Emilia Lecuona, Stephen F. Chiu, Qiang Wu, Melissa Querrey, Ramiro Fernandez, Félix L. Núñez-Santana, Haiying Sun, Sowmya Ravi, Chitaru Kurihara, James M. Walter, Nikita Joshi, Ziyou Ren, Scott C. Roberts, Alan Hauser, Daniel Kreisel, Wenjun Li, Navdeep S. Chandel, Alexander V. Misharin, Thalachallour Mohanakumar, G.R. Scott Budinger, Ankit Bharat

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Figure 8

ROS produced during ischemia reperfusion are responsible for TLR4 upregulation.

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ROS produced during ischemia reperfusion are responsible for TLR4 upregu...
(A) Human donor lungs being used for clinical transplantation were serially biopsied during the different phases of ischemia/reperfusion and development of ROS, and TLR4 transcription of flow-sorted donor TRAMs was determined. (B) WT BALB/c mice were transplanted with WT C57BL/6 donor lungs. In order to test the effect of ROS on TLR4 expression in TRAMs, we treated donors and recipient with NAC (100 mg/kg BW), MT (5 mg/kg BW, i.p.), or DM (10 mg/kg BW, i.v.), before and after reperfusion. Tlr4 mRNA levels were assessed 24 hours after ischemia by qPCR in TRAMs harvested from BALF before and after transplantation (n = 3–5). (C) Protein expression of TLR4 at the plasma membrane in TRAMs harvested as in B, assessed by flow cytometry (n = 4). (D) Immunocytochemistry for TLR4 of TRAMs harvested as described in B. Nr4a1–/– donor lungs, which do not develop PGD, were administered low-dose LPS and transplanted into WT recipients with or without NAC treatment. (E) Neutrophil infiltration was determined using flow cytometry and (F) lung function was determined using blood gases and P/F ratio. (B, C) Graphs were analyzed by 1-way ANOVA followed by Tukey’s post hoc test. (E, F) Graphs were analyzed by unpaired t test. *P < 0.05; ## and **P < 0.01; ### and ***P < 0.001; ****P < 0.0001. (A) *ROS; #TLR4 transcription. Scale bar: 10 μm. Arrow points to TLR4 at the plasma membrane.