Mitochondrial damage-associated molecular patterns released by lung transplants are associated with primary graft dysfunction

Abstract

Primary graft dysfunction (PGD) is a major limitation in short- and long-term lung transplant survival. Recent work has shown that mitochondrial damage-associated molecular patterns (mtDAMPs) can promote solid organ injury, but whether they contribute to PGD severity remains unclear. We quantitated circulating plasma mitochondrial DNA (mtDNA) in 62 patients, before lung transplantation and shortly after arrival to the intensive care unit. Although all recipients released mtDNA, high levels were associated with severe PGD development. In a mouse orthotopic lung transplant model of PGD, we detected airway cell-free damaged mitochondria and mtDNA in the peripheral circulation. Pharmacologic inhibition or genetic deletion of formylated peptide receptor 1 (FPR1), a chemotaxis sensor for N-formylated peptides released by damaged mitochondria, inhibited graft injury. An analysis of intragraft neutrophil-trafficking patterns reveals that FPR1 enhances neutrophil transepithelial migration and retention within airways but does not control extravasation. Using donor lungs that express a mitochondria-targeted reporter protein, we also show that FPR1-mediated neutrophil trafficking is coupled with the engulfment of damaged mitochondria, which in turn triggers reactive oxygen species (ROS)-induced pulmonary edema. Therefore, our data demonstrate an association between mtDAMP release and PGD development and suggest that neutrophil trafficking and effector responses to damaged mitochondria are drivers of graft damage.

Keywords: animal models; basic (laboratory) research/science; cellular biology; clinical research/practice; immunobiology; innate immunity; ischemia-reperfusion injury (IRI); lung (allograft) function/dysfunction; lung transplantation/pulmonology; mouse.

Figure 1.. Lung transplantation increase the levels of circulating Mt-DNA.

(A) Box and whiskers plot of circulating MT-CYB DNA levels obtained from lung recipients before lung transplantation and within 6 to 12 hours after ICU arrival. (B) Paired analysis plot of MT-CYB DNA levels for each patient before and after lung transplantation. (C) Box and whiskers plot of circulating Mt-CYTB DNA levels paired with qualitative variables for end-stage lung disease, gender and use of cardio pulmonary bypass (CBP). The dashed line represents the threshold limit for Mt-DNA detection (background). (**p< 0.01, *** p<0.001, ****p< 0.0001).

Figure 2.. High levels of circulating Mt-DNA are associated with severe PGD in lung transplant patients.

Box and whisker plots of circulating MT-CYB levels in relation to PGD scores at 72 hours after ICU arrival as represented (A) by separate PGD grades (**p< 0.01), (B) patients grouped together with PGD 0–1 grades versus patients with PGD grades 2 or 3 (*p< 0.05) or (C) patients grouped together with PGD 0–2 grades versus patients with PGD grade 3 (*p< 0.05). Error bars represent 95% confidence intervals for (A-C).

Figure 3.. Cell-free damaged mitochondria are released into the airways of lung transplant recipients.

(A) Representative H&E histopathology (N=5) of bronchiolar epithelium from B6 lung grafts and the corresponding right un-transplanted lung (Control) one day after reperfusion (200X magnification, red scale bar = 50 μm). (B) Flow cytometric (FACS) quantitation of (B) BALf cell-fee mitochondria and circulating plasma (C) MT-CYB from mouse lung transplant recipients 90 mins after reperfusion. Sham surgery was used as a control. Data shown as mean Mitotracker GreenFM⁺ events ± SEM and mean 1/Ct ± SEM where *p <0.05 (n = 5/group). (D) Representative transmission electron micrograph of cell-free mitochondria from the BAL of B6 lung recipients (N=5). Structures resembling mitochondrial cristae (red arrows) and a mitochondrial double membrane (white arrows) at 30 × 10³ X magnification where the black scale bar = 500 nM. An analysis of BALf cell-free graft- and recipient-derived mitochondria extracted from Dendra2 → B6 recipients. Tom22+ isolates were gated as in (E) to assess Dendra2 expression in (F) for control mitochondria isolated from Dendra2 lungs, (G) control mitochondria isolated from CD11b+ cells from the resting bone marrow of B6 mice and (H) BALf mitochondria isolated from Dendra2 → B6 recipients. Percent abundance of graft-derived mitochondria is shown as a mean ± SEM for 5 transplants. Measurement of inner mitochondrial membrane potential (Ψ_m) with the electrosensitive probe TMRM as represented by (I) histogram plot and (J) bar graph of freshly isolated mitochondria from B6 CD11b+ cells, resting Dendra2 lung tissue, cell-free mitochondria isolated from the BALf of Dendra2 > B6 lung recipients differentiated by Dendra2 expression. The result in (I) is a representative histogram of Ψ_m from 5 experiments and (J) is mean Ψ_m ± SEM where **p <0.01 (n = 5/group).

Figure 4.. The FPR1 inhibitor Cyclosporine H reduces signs of lung transplant-mediated IRI.

B6 recipients were treated daily with 50μg i.p. with the FPR1 inhibitor Cyclosporine H (Cy-H) for 3 consecutive days before receiving ischemically-injured B6 lungs. One day after reperfusion grafts were analyzed for signs of acute injury by (A) H&E histopathology (100X, scale bar 200 μm; representative of n=3/group) (B) lung injury score (n=3/group), (C) airway neutrophil accumulation quantified as (left panel) total number and (right panel) percent abundance relative to CD45⁺ cells (n=4/group) and (D) wet to dry ratio (n=4/group). (B-C) show means ± SEM, *p<0.05, **p<0.01.

Figure 5.. FPR1 expression in the recipient is sufficient to promote lung graft injury.

Lung grafts from indicated transplant combinations one-day after engraftment were assessed for signs of acute lung injury. (A) H&E stain (200X, red scale bar = 50μm, n=5/group). Red arrowheads indicate leukocyte infiltration within alveolar spaces. Histological based (B) leukocyte counts within airspaces (mean ± SEM, n=5/group) and (C) lung injury score (mean ± SEM, n=5/group). (D) Airway neutrophilia quantified by (left) total number and (right) percent abundance relative to CD45⁺ cells (mean ± SEM; n=5/group; *p<0.05, **p<0.01) in the BAL. (E) Lung transplant injury evaluated by (left panel) wet to dry weight ratio (mean ± SEM, n = 6/group) and (right panel) BALf protein content (mean ± SEM, n = 6/group).

Figure 6.. FPR1 promotes neutrophil migration into airspaces.

(A) B6 → B6 and B6 → FPR1^−/− recipients analyzed for neutrophil extravasation. PE-labeled Ly6G antibodies were injected intravenously into recipient mice 5 mins prior to euthanasia. Graft tissue was stained with Gr1-FITC antibodies allowing identification of intravascular neutrophils (Gr1-FITC⁺ Ly6G-PE⁺) and interstitial neutrophils (Gr1-FITC⁺ Ly6G-PE^-). Results shown are (left) representative contour plots from 4 transplants per group (right) or as a histogram denoting the ratio of graft intravascular to extravascular neutrophils as mean ± SEM (n=4/group; not significant; ns). (B) Transepithelial migration of B6 and FPR1 deficient neutrophils across mouse epithelial MLE12 monolayers in response to N-formylated peptides (fMLP; 1μM) and CXCL1 (10 ng/ml). Data shown as a mean ± SEM; 2 mice pooled per group; 3 independent experiments. Statistical differences between groups were determined by 2 way ANOVA with Sidack`s multiple comparison where p* <0.05, p ****<0.0001. (C) Neutrophil accumulation in non-airway tissue compartments. Indicated transplant recipients one day after reperfusion underwent BAL extraction prior to graft digestion and flow cytometry. Neutrophils quantified by total counts (upper panel) and (lower panel) percent abundance relative to CD45⁺ cells (mean ± SEM; *p<0.05, **p<0.01; n=5/group). (D) Neutrophils isolated from B6 LysM-EGFP and FPR1^−/− LysM-EGFP mice were introduced into the left main bronchus of ischemically-injured B6 donor lungs and immediately transplanted into B6 recipients. Lung recipients were euthanized 90 minutes after reperfusion and EGFP⁺ neutrophil retention within the airway (BAL), trafficking into non-airway graft tissues and peripheral blood were analyzed by FACS. Representative contour plots of EGFP⁺ neutrophil percent abundance for indicated tissue compartments from 5 transplants per group. (E) Histogram shows mean number of EGFP⁺ neutrophils retained within airways (n =5 / group; * p<0.05).

Figure 7.. Neutrophils that engulf damaged mitochondria induce ROS-mediated pulmonary edema.

(A) BAL neutrophils from Dendra2 > B6 and Dendra2 > FPR1^−/− recipients were assessed for uptake of Dendra2 containing mitochondria from lung grafts 3 hours after transplant. Left contour plots show representative percent abundance and the right panel depicts corresponding mean fluorescence intensities (MFI) for 5 transplants (**p<0.01). (B) BAL neutrophils gated on indicated Dendra2+ and Dendra2- gates, as represented in (A), were probed for cellular ROS with CellROX. Data shown are representative FACS histograms from 5 transplants per group. B6 and FPR1^−/− neutrophils were co-cultured with indicated amounts of damaged mitochondria prepared from Dendra2 lungs for 20 minutes and then assessed for (C, left panel) percent engulfment, (C, right panel) engulfment per cell, and (D) ROS production. Results shown are a representative experiment (n=4/measurement; **p< 0.01, ***p< 0.001) from 3 independently conducted experiments. (E) 10⁶ B6 and FPR1^−/− neutrophils co-cultured with 25 μg/ml (Mt-DAMPs²⁵) or 100 μg/ml (Mt-DAMPs¹⁰⁰) of damaged mitochondria for 20 mins in the presence or absence of the ROS inhibitor DPI (10 μM), washed twice and administered down the trachea of resting B6 mice. Pulmonary edema was evaluated 18 hours later by wet to dry weight ratio (N=5/group where p** < 0.01, ****p< 0.0001). Data in (A) and (C-E) are shown with means ± SEM.

Figure 8.. Mt-DAMP release by lung transplants exacerbates neutrophil-mediated tissue injury.

(A) Ischemia-reperfusion injury promotes necrotic cell release of damaged mitochondria, which in turn emit Mt DAMPs. (B) Neutrophil transepithelial migration into air spaces is driven by FPR1-mediated chemotaxis towards mitochondrial N-formylated peptides. (C) While in the airspaces, neutrophils engulf damaged mitochondria that leads to ROS production that compromises homeostatic barriers allowing further leakage of Mt-DAMPs, such as Mt-DNA, into the peripheral circulation.