DAP12 expression in lung macrophages mediates ischemia/reperfusion injury by promoting neutrophil extravasation

Abstract

Neutrophils are critical mediators of innate immune responses and contribute to tissue injury. However, immune pathways that regulate neutrophil recruitment to injured tissues during noninfectious inflammation remain poorly understood. DAP12 is a cell membrane-associated protein that is expressed in myeloid cells and can either augment or dampen innate inflammatory responses during infections. To elucidate the role of DAP12 in pulmonary ischemia/reperfusion injury (IRI), we took advantage of a clinically relevant mouse model of transplant-mediated lung IRI. This technique allowed us to dissect the importance of DAP12 in tissue-resident cells and those that infiltrate injured tissue from the periphery during noninfectious inflammation. Macrophages in both mouse and human lungs that have been subjected to cold ischemic storage express DAP12. We found that donor, but not recipient, deficiency in DAP12 protected against pulmonary IRI. Analysis of the immune response showed that DAP12 promotes the survival of tissue-resident alveolar macrophages and contributes to local production of neutrophil chemoattractants. Intravital imaging demonstrated a transendothelial migration defect into DAP12-deficient lungs, which can be rescued by local administration of the neutrophil chemokine CXCL2. We have uncovered a previously unrecognized role for DAP12 expression in tissue-resident alveolar macrophages in mediating acute noninfectious tissue injury through regulation of neutrophil trafficking.

Figure 1. DAP12-deficiency on donor cells attenuates transplant-mediated lung ischemia reperfusion injury

A. Arterial pO2 levels six hours after syngeneic lung transplantation, (B6 WT→B6 WT, n=14; B6 DAP12KO→B6 WT, n=8; B6 WT→B6 DAP12KO, n=7, DAP12KO→DAP12KO, n=5) *p<0.05 (One-way ANOVA with Tukey’s post-test). B. DAP12 qPCR on donor-derived alveolar macrophages and endothelial cells sorted two hours after B6 WT CD45.2→ B6 WT CD45.1 or B6 DAP12KO CD45.2 → B6 WT CD45.1 lung transplantation. Results were normalized to β₂-microglobulin and represent four separate experiments, *p<0.05 (paired t test). C. Immunofluorescence staining of ischemic lungs. Top panel, B6 WT, middle panel, B6 DAP12KO, and bottom panel human lung tissue after cold ischemic storage, immediately prior to transplantation and two hours after reperfusion. Tissue was stained for DAP12 (green), CD68 (red), and DAPI (blue) (20×).

Figure 2. DAP12KO bone marrow-derived macrophages (BMDM) have decreased survival and cytokine/chemokine production upon incubation with lung transplant lysate in vitro

A. B6 WT and B6 DAP12KO BMDM viability following 17 hours of incubation with B6 WT → B6 WT transplant lysate demonstrated as percentage Annexin V⁺7AAD⁺. Graph is representative of four separate experiments performed in duplicates with similar results. B. Representative dot plots of B6 WT and B6 DAP12KO BMDM after 17 hours of incubation with transplant lysate. C. BMDM production of cytokines and chemokines as measured by ELISA following 17 hours of incubation with B6 WT → B6 WT transplant lysate. IL-6, CXCL1 and CXCL2 were significantly decreased in DAP12KO BMDM in two out of three independent experiments. Graph represents one experiment repeated independently three times performed at least in duplicate +/−SEM (*p<0.05, **p<0.01, two-way ANOVA).

Figure 3. Lower abundance of DAP12KO donor-derived macrophages and decreased expression of cytokines/chemokines following pulmonary ischemia-reperfusion injury in vivo

A. Representative dot plots of donor-derived alveolar macrophages (CD64⁺CD11c⁺SiglecF⁺CD11b⁻) in graft tissue 2 hours after B6 WT CD45.2 → B6 WT CD45.1 or B6 DAP12KO CD45.2 → B6 WT CD45.1 lung transplantation. Cells were first gated on CD45.2⁺CD45.1⁻ cells as seen in Supplemental Figure 1. B. Graphical representation of donor macrophage numbers per mg of tissue (n=7 per experimental group). Based on paired t test, *p<0.05. C. Expression of cytokines and chemokines in donor-derived, sorted alveolar macrophages (CD45.2⁺CD45.1⁻CD64⁺CD11c⁺SiglecF⁺CD11b⁻) (n=4 per experimental group) was assessed by qPCR. Results were normalized to β2-microglobulin and compared to the WT population in each experiment. Based on two-way ANOVA, **p<0.01, *p<0.05.

Figure 4. Recipients of DAP12KO lungs have decreased graft infiltration of neutrophils

A. Representative dot plots of neutrophil gating in B6 WT CD45.2 → B6 WT CD45.1 lung grafts (n=7 per experimental group). Neutrophils were gated on recipient CD45.2CD45.1⁺ cells and identified as Ly6G⁺Gr1⁺. These cells were CD11b^hi and had high side scatter. B. Representative dot plot showing percentage of extravasated neutrophils in B6 WT CD45.2 → B6 WT CD45.1 or B6 DAP12KO CD45.2 → B6 WT CD45.1 and assessed based on their staining for PE-labeled Ly6G, which was injected intravenously into recipient mice prior to sacrifice. Intravascular neutrophils were Ly6G-PE⁺ Ly6G-APC⁺ while interstitial neutrophils were Ly6G-PE⁻ Ly6G-APC⁺. C. Graphical representation of absolute numbers of neutrophils (left) and neutrophil extravascular (EV) vs. intravascular (IV) distribution (right). Graphs represent seven separate experiments. Based on paired t test, **p<0.01.

Figure 5. Neutrophil transendothelial migration in DAP12KO lung grafts is rescued by endobronchial administration of WT BMDM or CXCL2

A. Intravital 2P microscopy was initiated three hours after transplantation into LysM-GFP hosts. Snapshots of time points immediately after start of imaging and 45 minutes later showing B6 WT and B6 DAP12KO grafts as well as B6 DAP12KO lungs that received WT B6 BMDM or recombinant CXCL2 endobronchially immediately prior to reperfusion (Supplemental Movies 1–8). Green-LysM-GFP⁺ neutrophils, red-non-targeted quantum dots, blue-second harmonic generation signal. Bar = 50 µm. B. Graphical representation of neutrophil numbers per mm³ at 0, 30, and 45 minutes after initiation of imaging (n=3–4 transplants per experimental group with 2–5 areas analyzed per transplanted lung). No statistically significant differences were detected between the 4 experimental groups at any time point. C. Graphical representation of percentage of extravasated neutrophils at 0, 30, and 45 minutes after initiation of imaging (n=3–4 transplants per experimental group with 2–5 areas analyzed per transplanted lung). Each dot represents a separate area of the lung graft. (0 min, DAP12KO vs WT, DAP12KO vs. DAP12KO + WT BMDM or DAP12KO + CXCL2, p<0.05; 30 min, DAP12KO vs. WT, DAP12KO + WT BMDM or DAP12KO + CXCL2, p<0.05; 45 min, DAP12KO vs. WT, DAP12KO + WT BMDM or DAP12KO + CXCL2, p<0.05). Data were analyzed with a two-way ANOVA with Tukey’s post-test.

Figure 6. Treatment of DAP12KO lung grafts with DAP12KO BMDM or PBS

A and D. Intravital 2P microscopy was initiated three hours after transplantation into LysM-GFP hosts. Snapshots of time points immediately after start of imaging and 45 minutes later showing B6 DAP12KO grafts that received DAP12KO B6 BMDM (A) or PBS (D) endobronchially immediately prior to reperfusion. Green-LysM-GFP⁺ neutrophils, red-non-targeted quantum dots, blue-second harmonic generation signal. B and E. Graphical representation of neutrophil numbers per mm³ at 0, 30, and 45 minutes after initiation of imaging (n=3–4 transplants per experimental group with 2–5 areas analyzed per transplanted lung). No statistically significant differences were detected between the 4 experimental groups at any time point. C and F. Graphical representation of percentage of extravasated neutrophils at 0, 30, and 45 minutes after initiation of imaging (n=3–4 transplants per experimental group with 2–5 areas analyzed per transplanted lung). Each dot represents a separate area of the lung graft. (0 min, DAP12KO vs DAP12KO +DAP12KO BMDM, DAP12KO + WT BMDM vs DAP12KO + DAP12KO BMDM, p<0.05; 30 min, B6 vs DAP12KO +DAP12KO BMDM, DAP12KO vs DAP12KO +DAP12KO BMDM, DAP12KO +WT BMDM vsDAP12KO +DAP12KO BMDM, p>0.05; 45 min, B6 vs DAP12KO +DAP12KO BMDM, DAP12KO vs DAP12KO +DAP12KO BMDM, DAP12KO +WT BMDM vs DAP12KO +DAP12KO BMDM, p<0.05) (0min, B6 vs DAP12KO +PBS, DAP12KO +CXCL2 vs DAP12KO +PBS, p<0.05; 30 min, B6 vs DAP12KO +PBS, DAP12KO +CXCL2 vs DAP12KO +PBS, p<0.05; 45 min, B6 vs DAP12KO +PBS, DAP12KO +CXCL2 vs DAP12KO +PBS, p<0.05). Data were analyzed with a two-way ANOVA with Tukey’s post-test.