30.06 Donor-derived intravascular TLR-dependent Ly6C^low monocytes lead primary lung allograft dysfunction

Z. Zheng¹, S. Chiu¹, H. Sun¹, Z. J. Zhang¹, M. M. DeCamp¹, D. Kreisel², S. Budinger¹, H. Perlman¹, A. Misharin¹, A. Bharat^{1 1}Northwestern University,Surgery/ Thoracic,Chicago, IL, USA ²Washington University,St. Louis, MO, USA

Introduction: Primary graft dysfunction (PGD) affects over 50% of lung recipients and remains the predominant cause of short- and long-term mortality. Neutrophil infiltration into the allograft mediates PGD. However, the mechanisms that lead to neutrophil trafficking remain unknown. Here we demonstrate that a unique subset of intravascular monocytes, that cannot be flushed using current preservative techniques, leads to PGD following lung transplantation.

Methods: Murine allogeneic left lung transplant was used for these studies. Neutrophil infiltration was determined using intravital 2-photon microscopy and flow-cytometry. Graft function was determined using arterial oxygen after clamping the right hilum for 5 minutes. Intravenous clodronate loaded liposomes (Clo-lip) was used to induce apoptosis in circulating monocytes.

Results: Perfused murine lungs demonstrated intravascular CD11b⁺MHCII^–CD64^+/-Ly6C^low monocytes (Ly6C^lowM) that constituted ~3% of resident CD45⁺ cells. These Ly6C^lowM shared functional phenotype with CD14⁺CD16⁺ monocytes detected in perfused human lungs. Pre-treatment of donors with Clo-lip depleted Ly6C^lowM in donor lungs (<0.3%). Following murine allogeneic left lung transplant, there was a robust neutrophil infiltration on intravital 2-photon imaging (Fig 1A) that was prevented by a single dose of Clo-lip in the donors. Donor Clo-lip treatment resulted in preserved allograft architecture and oxygenation at 24 hours (Fig 1B&C). Treatment with anti-CCR2 antibodies which selectively deplete Ly6C^high monocytes, but not Ly6C^lowM, worsened neutrophilia (3 folds) and graft function. Homozygous deletion of CX₃CR1 chemokine receptor resulted in absent Ly6C^lowM in the lungs without affecting circulating Ly6C^lowM, indicating a role of CX₃CR1 binding to its ligand CX₃CL1 expressed on endothelium. CX₃CR1^–/^– donor lungs demonstrated suppressed neutrophilia (2.5 fold reduction) and preserved function. The CX₃CR1-CX₃CL1 covalent bonds were calcium dependent and were neutralized by ethylene glycol tetraacetic acid (EGTA) resulting in flushing of Ly6C^lowM during perfusion. Reconstitution of Clo-lip treated donor lungs with MyD88-TRIFF knockout Ly6C^lowM, with deficient Toll signaling, failed to restore allograft neutrophilia, in contast to wild-type Ly6C^lowM.

Conclusion: Pulmonary intravascular Ly6C^lowM are a novel subset of monocytes that cannot be flushed using standard preservative techniques. Donor Ly6C^lowM, transplanted along with the donor lungs, are activated through TLR pathway mediating neutrophil trafficking and PGD. Ly6C^lowM are bound to the donor endothelium using covalent bonds that can be neutralized using a calcium chelator. Depletion of these cells in the donor prevented PGD following lung transplantation.