30.03 Donor-derived Intravascular Monocytes Lead to Native Lung Neutrophilia During Single Lung Transplant

S. Chiu^1,4, Z. Zheng⁴, H. Sun³, R. Fernandez¹, M. DeCamp¹, M. Abecassis⁴, D. Kreisel⁶, G. S. Budinger³, H. Perlman⁵, A. Misharin^3,5, A. Bharat^{1 1}Northwestern University,Thoracic Surgery,Chicago, IL, USA ³Northwestern University,Pulmonary And Critical Care Medicine,Chicago, IL, USA ⁴Northwestern University,Kovler Comprehensive Transplant Center,Chicago, IL, USA ⁵Northwestern University,Rheumatology,Chicago, IL, USA ⁶Washington University,Thoracic Surgery,St. Louis, MO, USA

Introduction: Acute deterioration of lung function due to native lung pneumonitis (NLP) following single lung transplant occurs in up to 10% of patients. Prior studies have shown that neutrophilia leads to NLP. However, it remains unclear why native lungs would develop neutrophilia without any surgical manipulation. Here we show that a unique subset of intravascular monocytes in the donor lungs, which cannot be flushed using current preservation techniques, migrates to the native lung and causes NLP.

Methods: Allogeneic single left lung transplant was performed utilizing wild-type (WT) C57BL/6J (B6) CD45.1 donor lungs and Balb/cJ (Bc) CD45.2 recipients as control. In the experimental arms, two sets of donors were used: (1) B6-WT donors treated with intravenous Clodronate-loaded liposomes (Clo-lip) to deplete intravascular Ly6C^low monocytes, and (2) B6-CX₃CR1^-/- (knockout) donor lungs which were deficient in chemokine receptor CX₃CR1 function and therefore had deficient Ly6C^low monocyte-endothelial cell binding. At 24 hours post-transplant, native right lungs were isolated and prepared for flow cytometric analysis. A sequential gating strategy was used to identify Ly6G⁺ neutrophils and CD11b⁺MHCII^–CD64^+/-Ly6C^low monocytes and cell counts performed. Student’s t-test was used to compare the means.

Results: Donor lungs perfused with current preservative solution demonstrated presence of intravascular CD11b+MHCII^–CD64^+/-Ly6C^low monocytes (3% of resident CD45+ cells). Using CD45.1 donors and CD45.2 recipients, we determined that 8% of the Ly6C^low monocytes in the native lung were donor-derived at 24 hours. Native lungs demonstrated 2.5 fold increased neutrophils following single allogeneic lung transplantation compared to sham operated mice. Donor treatment with single intravenous injection of Clo-lip depleted these monocytes in the donor lungs (<0.5% of CD45+ cells). Further, in recipients of Clo-lip treated donors, no donor-derived monocytes were found in the native lungs at 24 hours. Donor Clo-lip treatment abrogated native lung neutrophilia (n=7, p=0.03). CX₃CR1 knockout donor mice had decreased Ly6C^low monocytes in the pulmonary intravascular space (<0.5%) and allogeneic lung transplant from CX₃CR1 knockout mice revealed suppressed neutrophilia in the native lungs, similar to donor Clo-lip treatment.

Conclusions: Donor-derived pulmonary intravascular Ly6C^low monocytes migrate to the contralateral native lung following single lung transplantation and can induce neutrophilia upon reaching the native lungs. The mechanisms that lead to the activation of these monocytes as well migration pathways would provide insight into native lung pneumonitis.