Crosstalk between nonclassical monocytes and alveolar macrophages mediates transplant ischemia-reperfusion injury through classical monocyte recruitment

Abstract

Primary graft dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular nonclassical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that depletion of donor NCM abrogated CM recruitment, single cell RNA sequencing identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine KOs and chimeras indicated that IL-1β production by donor NCM was responsible for the early activation of AM and CCL2 release. IL-1β production by NCM was NLRP3 inflammasome dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL-1R-dependent activation of the PKC and NF-κB pathway. Accordingly, we show that IL-1β-dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL-1β, or IL-1R antagonism and inflammasome inhibition abrogated recruitment of CM and PGD and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.

Keywords: Organ transplantation; Transplantation.

Conflict of interest statement

Conflict of interest: DK has a pending US patent entitled “Compositions and methods for detecting CCR2 receptors” (application no. 15/611,577).

Figures

Figure 1. The CCL2-CCR2 axis is necessary for recipient classical monocytes (CM) recruitment to the allograft.

(A) Flow cytometry quantification of CM (live CD45+Ly6G–NK1.1–CD11b+SiglecF–CD24–Ly6Chi) recruited into the allograft after treatment of recipients with i.v. IgG isotype or anti-CCR2 antibodies (n = 5). (B) Flow cytometry quantification of CM as described in A, recruited into the allograft using WT or Ccr2–/– recipient mice (n = 5). (C) Flow cytometry quantification of CM (live CD45+Ly6G–NK1.1–CD19–CD11b+CF4/80–CD11c–Ly6Chi) in spleens of WT or Ccr2–/– recipients after lung transplant (n = 5–6). (D) Flow cytometry quantification of CM as described in A, recruited into the allograft after treatment of recipients with i.v. IgG isotype or anti-CCL2 antibodies in recipient mice (n = 3). Graphs show means ± SD. Graphs were analyzed by unpaired Student’s t test. ***P < 0.001; ****P < 0.0001.

Figure 2. Depletion of donor nonclassical monocytes (NCM) suppresses the recruitment of recipient splenic classical monocytes (CM) to the allograft.

(A–C) Intravital 2-photon imaging between 2 and 2.5 hours after reperfusion. (A) Representative still images of WT and Nr4a1–/– donor grafts. Green, CCR2-GFP; red, Qdot655 blood vessels. (B and C) CCR2-GFP cell density (B) and percent of extravasated CCR2-GFP (C) calculated using NIH ImageJ software in WT and Nr4a1–/– mice grafts after transplantation. (D) Flow cytometry quantification of CM (live CD45+Ly6G–NK1.1–CD11b+SiglecF–CD24–Ly6Chi) recruited into the allograft after i.v. injection of PBS liposomes (PBS-lip) or clodronate liposomes (Clo-lip) in donor mice or using WT or Nr4a1–/– as donor lungs (n = 5). (E) Normalized counts per minute (CPM) of Ccl2 in sorted donor NCM isolated from allografts 2 and 24 hours after transplant. (F) Relative Ccl2 mRNA levels of human and mouse NCM isolated before and after reperfusion (n = 3). (G) CCL2 levels in blood from mice in D (n = 5). Graphs show means ± SD. The graph in B was analyzed by 2-way ANOVA followed by Sidak’s post hoc test. *WT versus Nr4a1–/–; #WT 2 hours versus WT 2.5 hours. The graph in E was analyzed by 1-way ANOVA followed by Tukey’s post hoc test. Graphs in C, D, F, and G were analyzed by unpaired Student’s t test. #P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bar: 50 μm.

Figure 3. Depletion of donor alveolar macrophages (AM) suppresses recruitment of recipient classical monocytes (CM) to the transplanted lung.

(A) Flow cytometry showing percentage of cells of donor (CD45.1) and recipient (CD45.2) origin in the allograft 24 hours after transplantation. Neutrophils were gated as live CD45+Ly6G+CD11b+CD24+SSChi; AM were gated as live CD45+Ly6G–NK1.1–SiglecF+CD64+CD11c+; CM were gated as live CD45+Ly6G–NK1.1–CD11b+SiglecF–CD24–Ly6Chi; NCM were gated as live CD45+Ly6G–NK1.1–CD11b+SiglecF–CD24–Ly6Clo (n = 3); and interstitial macrophages (IM) were gated as live CD45+Ly6G–NK1.1–CD11b+MHC II+CD11c+CD64+CD24− (n = 3–6). (B) UMAP plot (left) and feature plots (middle and right) showing specific cell populations and expression of Ccl2 in naive lungs and in allografts (Allo) 24 hours after transplant. (C) Flow cytometry showing percentage of RFP+ cells in lung allografts after transplantation of donor Ccl2-rfp grafts into WT recipient. AM and NCM were gated as in A. (D) Normalized counts per minute (CPM) of Ccl2 in sorted mouse AM isolated from allografts 2 hours after transplant (n = 4). (E) Relative Ccl2 mRNA levels of human and mouse AM isolated before and after reperfusion (n = 3–5). (F) Flow cytometry quantification of CM gated as in A, recruited into the allograft after intratracheal administration of PBS liposomes (PBS-lip) or clodronate liposomes (Clo-lip) in the donor mice (n = 4). (G) Flow cytometry quantification of extravasated neutrophils in the allograft gated as in A, after intratracheal administration of PBS-lip or Clo-lip in donor mice (n = 3). (H) Relative Ccl2 mRNA levels in AM isolated from WT, Cd169Cre, Ccl-rfpfl/fl, and Cd169CreCcl2-rfpfl/fl allografts and compared with naive AM (n = 5). (I) Blood CCL2 levels after transplant combinations described in H (n = 5). (J) Flow cytometry quantification of CM gated as in A, after transplant combinations described in H (n = 5). Graphs show means ± SD. Graphs in C–G were analyzed by unpaired Student’s t test. Graphs in H–J were analyzed by 1-way ANOVA followed by Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4. IL-1β is necessary for donor alveolar macrophages (AM) to produce CCL2.

(A) AM were isolated from WT or Il1r–/– mice and incubated in vitro with PBS or 1 ng/μL of mouse recombinant IL-1β. Ccl2 mRNA expression was measured by qPCR 24 hours after incubation (n = 6–8). (B) AM isolated from WT mice were incubated for 30 minutes in vitro with DMSO (control), 50 μM LY294002 (LY), 10 μM Bisindolylmaleimide-I (Bis), or 1 μM BAY11-7082 before adding 1 ng/μL of mouse recombinant IL-1β. Ccl2 mRNA expression was measured by qPCR 24 hours after incubation (n = 3–7). (C) Donor AM were isolated from WT or Il1r–/– allografts 24 hours after transplant, and Ccl2 mRNA expression was measured by qPCR (n = 4–5). (D) Flow cytometry quantification of CM (live CD45+Ly6G–NK1.1–CD11b+SiglecF–CD24–Ly6Chi) from allografts harvested at the indicated times from experiments in which donor lungs were treated with PBS or anakinra, or in which recipients were treated with isotype control and anti–IL-1β antibody (n = 3 per group). Graphs show means ± SD. Graphs in A–C were analyzed by unpaired Student’s t test. Graph in D was analyzed by 2-way ANOVA, followed by Sidak’s post hoc test. *PBS versus anakinra; #isotype versus anti–IL-1β antibody. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ###P < 0.001; ####P < 0.0001.

Figure 5. Nonclassical monocyte–derived (NCM-derived) IL-1β promotes CCL2 production by donor alveolar macrophages (AM).

(A) UMAP plot (left) and feature plots (middle and right) showing specific cell populations and expression of Il1β in naive lungs and in allografts (Allo) 24 hours after transplantation. (B) Ccl2 mRNA levels determined by qPCR in AM from the allograft after i.v. administration of IgG isotype or anti-Ly6G antibodies in recipient mice (n = 2–3). (C) Relative Il1β mRNA levels of mouse donor NCM isolated before and after reperfusion (n = 3–4). (D) Ccl2 mRNA levels determined by qPCR in AM isolated WT or Nr4a1–/– lung allografts 24 hours after transplantation (n = 4–6). (E) Flow cytometry quantification of CM (live CD45+Ly6G–NK1.1–CD11b+SiglecF–CD24–Ly6Chi) recruited into the allograft after treatment of recipients with i.v. IgG isotype or anti-CXCL2 antibodies (n = 5). Graphs show means ± SD. Graphs were analyzed by unpaired Student’s t test.*P < 0.05.

Figure 6. Nonclassical monocytes (NCM) produce IL-1β via NLRP3 inflammasome activation.

(A) Ccl2 mRNA levels determined by qPCR in AM isolated from WT or Nlrp3–/– lung allografts 24 hours after transplantation (n = 3). (B) Flow cytometry quantification of CM (live CD45+Ly6G–NK1.1–CD11b+SiglecF–CD24–Ly6Chi) recruited to allografts from WT, Nlrp3–/–, or Nlrp3–/– reconstituted with WT NCM donor mice (n = 3–4). (C) Flow cytometry quantification of neutrophils (live CD45+Ly6G+CD11b+CD24+SSChi) recruited into WT of Nlrp3–/– allografts 24 hours after transplantation (n = 5–7). (D) Flow cytometry quantification of CM gated as in C, recruited into the allograft after donor treatment with 50 μg/g body weight glyburide (n = 4). (E) Flow cytometry quantification of neutrophils gated as in B, recruited into the allograft after i.p. administration glyburide in the donor mice (n = 4–7). (F) Flow cytometry quantification of extravasated neutrophils in the allograft gated as in B, after donor treatment with glyburide (n = 4). Graphs show means ± SD. Graph in C was analyzed by 1-way ANOVA, followed by Tukey’s post hoc test. All other graphs were analyzed by unpaired Student’s t test. *P < 0.05; ***P < 0.001; ****P < 0.0001.