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 Transplantation

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 4

Influx of recipient neutrophils into the allograft containing LPS is abrogated by depletion of donor TRAMs and is CXCL2 dependent.

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Influx of recipient neutrophils into the allograft containing LPS is abr...
(A) Diagram depicting experiment shown in B. WT C57BL/6 mice received BALB/c allogeneic donor lungs containing low-dose LPS (0.5 μg/g BW) injected i.t., insufficient to cause native lung injury. To deplete NCMs, donors were treated with i.v. Clo-lip before i.t. LPS. To deplete both donor monocytes and donor TRAMs, we treated the donors with i.v. Clo-lip and i.t. Clo-lip, respectively, before i.t. LPS. Next, we used Nr4a1–/– donor lungs containing low-dose LPS and transplanted them into WT recipients after depletion of TRAMs using i.t. Clo-lip, or using PBS-lip as control. Last, to determine whether CXCL2 secretion by donor TRAMs led to neutrophil recruitment, we treated recipients of Nr4a1–/– donor lungs containing low-dose LPS with anti-CXCL2 antibodies at the time of transplantation. Additionally, we transplanted Nr4a1–/– donor lungs containing low-dose LPS into Cxcr2–/– recipients and determined neutrophil infiltration at 24 hours.(B) Quantification of neutrophil infiltration into lungs after these treatments was performed using flow cytometry (n = 4–5). (C) CXCL2 chemokine levels in BALF of recipient mice after receiving low-dose LPS-treated Nr4a1–/– donor lungs compared with PBS-treated Nr4a1–/– donor lungs (n = 5). (D) TRAMs were isolated from endotoxin-negative and endotoxin-positive human donor lungs at 120 minutes after reperfusion and analyzed for CXCL2 transcription (n = 4). All graphs show mean ± SD. Graph in B was analyzed by 1-way ANOVA followed by Tukey’s post hoc test. Graphs in C and D were analyzed by unpaired t test. ***P < 0.01; ****P < 0.0001. CT, control.