Purpose: Neutrophils mediate primary lung allograft dysfunction (PGD), the predominant cause of early graft loss following lung transplant. The mechanisms of neutrophil infiltration remain incompletely characterized. We show that donor-derived nonclassical endothelial-bound CX₃CR1⁺CCR2^–Ly6C^low monocytes (Ly6C^lowM) mediate neutrophil infiltration and PGD.

Methods: Allogeneic single lung transplant was done using wild-type (WT) or CX₃CR1 knockout (KO) mice. Intravascular (IV) clodronate liposomes (Clo-lip) were used to deplete all monocytes while anti-CCR2 was used to selectively deplete classical CCR2⁺Ly6C^hi monocytes. Neutrophil infiltration was analyzed by intravital 2-photon microscopy and multichannel flow cytometry. PGD was determined by arterial blood gas and histology.

Results: Intravascular Ly6C^lowM represented 2-5% of lung myeloid cells in donor lungs and persisted despite standard flushing techniques. IV Clo-lip eliminated all Ly6C^lowM without affecting lung-resident macrophages, dendritic cells, or interstitial classical monocytes. Allograft neutrophil infiltration was abrogated by depletion of Ly6C^lowM in donors (Fig 1A). At 24 hours, there was a 2-fold reduction in BAL neutrophils (Control: 3.0±0.8×10⁵ v. Clo-lip: 1.4±0.3×10⁵, n=8) and protection against PGD (Fig 1B&1C). Donor lungs reconstituted with WT, but not MyD88-TRIF KO Ly6C^lowM, which lack TLR signaling, restored allograft neutrophil infiltration. CX₃CR1 deletion in donors led to decreased circulating Ly6C^lowM and 2-fold decreased neutrophils (CX₃CR1 KO 0.7±0.2×10⁶ v. WT 1.3±0.2×10⁶, n=4). Depletion of donor classical monocytes with anti-CCR2 had no effect on neutrophil infiltration (Control: 1.4±0.1×10^6 v. anti-CCR2: 1.9±0.5×10^6, n=6).

Conclusion: Donor-derived nonclassical endothelial-bound Ly6C^lowM in the lung are a novel subset of monocytes that are dependent on TLR signaling and mediate PGD. Depletion of donor lung Ly6C^lowM abrogates PGD. Donor nonclassical monocytes are an attractive target for intervention to prevent PGD during lung transplant.

CITATION INFORMATION: Chiu S, Zheng Z, Sun H, DeCamp, Jr M, Budinger G, Perlman H, Misharin A, Bharat A. Donor-Derived Toll-Like Receptor-Dependent Intravascular Monocytes Cause Primary Lung Graft Dysfunction. Am J Transplant. 2016;16 (suppl 3).

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