Spleen-derived classical monocytes mediate lung ischemia-reperfusion injury through IL-1β

Abstract

Ischemia-reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short-term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that CCR2+ classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia-reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88-dependent IL-1β production by graft-infiltrating classical monocytes, which downregulated the expression of the tight junction-associated protein ZO-2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia-reperfusion injury in the clinic.

Keywords: Cellular immune response; Organ transplantation; Transplantation.

Conflict of interest statement

Conflict of interest: YL, DK, and KJL have a pending US patent entitled “Compositions and methods for detecting CCR2 receptors” (application number 15/611,577).

Figures

Figure 1. Classical monocytes recruited to the lung after IRI mediate neutrophil extravasation.

WT mice underwent IRI using hilar clamping, and the myeloid cell populations were analyzed by flow cytometry. (A) Representative flow cytometry plots (left) and quantification (right) of extravasated neutrophils and recruited classical monocytes (CM) in resting state or that of hilar clamp–mediated IRI are demonstrated. Neutrophils and classical monocytes were gated on live CD45+Ly6G+CD11b+SSChi and live CD45+Ly6G–NK1.1–SiglecF–CD11b+CD64–Ly6Chi cells, respectively. (B) Correlation between recruitment of classical monocytes following reperfusion of human lung allografts and increase in neutrophils in the BALF. The ratio of classical monocytes normalized by alveolar macrophages was correlated with an increase in bronchoalveolar neutrophils before and 15 minutes following reperfusion. Pearson’s correlation coefficient (r) was significant. (C) The percentage of extravasated neutrophils was determined after hilar clamp–mediated IRI in mice that were systemically depleted of monocytes 24 hours prior to IRI using Clo-lip (left), DT (CD11b-DTR mouse; middle), or anti-CCR2 antibody treatment (right). Data are expressed as median with interquartile range. n = 5–13 per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Mann-Whitney U test (A and C).

Figure 2. Classical monocytes mediating neutrophil extravasation in lungs subjected to IRI through hilar clamping are mobilized from the spleen.

Numbers of classical monocytes in the (A) lung, (B) spleen, and (C) bone marrow before and 2 hours after hilar clamp–mediated lung IRI. Numbers of classical monocytes in the lung and spleen are shown after treatment with isotype control or anti-CCR2 antibody. (D) Numbers of classical monocytes in bone marrow of resting mice after treatment with isotype control or anti-CCR2 antibody. Data are expressed as median with interquartile range. n = 5 per group. *P < 0.05, Mann-Whitney U test. (E) Recruitment of classical monocytes to injured lung and (F) percentage of extravasated neutrophils in injured lung after hilar clamp–mediated IRI in the presence or absence of a spleen. Congenic spleen transplants were performed into hosts that had undergone native splenectomies. Splenectomy of the transplanted spleen (resplenectomy) rendered these mice asplenic. txp, transplant. For E, 1 statistical outlier in the spleen transplant and 1 statistical outlier in resplenectomy group were excluded from the analysis. For F, 1 statistical outlier was excluded in the splenectomy group. Data are expressed as mean ± SEM. n = 6–8 per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA with post hoc Holm-Šídák test (A–C, E, F).

Figure 3. Classical monocytes are mobilized from the recipient’s spleen to lung grafts after transplantation.

(A) Representative contour plots and histograms depict recipient (CD45.2+) CD11b+Ly6chiCCR2+ monocytes in pulmonary grafts and peripheral blood 2 hours after transplantation of B6 CD45.1+ lungs into control or splenectomized congenic B6 CD45.2+ mice. (B) Graphic presentation of monocytes as percentages of recipient CD45.2+ hematopoietic cells and absolute numbers in lung grafts and peripheral blood. Data are expressed as median with interquartile range. n = 4 per group. *P < 0.05, unpaired t test. (C) A CCR2-specific PET radiotracer (64Cu-DOTA-ECL1i) was injected into control (left) or splenectomized (right) B6 recipients of syngeneic lung grafts 2 hours after transplantation and imaged by 0- to 60-minute dynamic PET/CT scanning after intravenous injection. L, liver; K, kidney; B, bladder. Circle shows lung graft. (D) Graphic presentation of 64Cu-DOTA-ECL1i uptake in native right lungs and pulmonary grafts 2 hours after transplantation into control or splenectomized syngeneic B6 recipients. Data are expressed as mean ± SEM. n = 4 per group. *P < 0.05; **P < 0.01; ****P < 0.0001, 1-way ANOVA with post hoc Holm-Šídák test. (E) Depletion of nonclassical monocytes in B6 donor lungs through Clo-lip or genetic deletion of NR4A1 led to a reduction in the serum MCP-1 levels of the BALB/c recipient 2 hours after reperfusion compared with control PBS-liposome treatment mice. (F) Recruitment of BALB/c recipient classical monocytes into the B6 allograft was significantly reduced 2 hours after reperfusion when donor lungs were treated with Clo-lip to deplete donor nonclassical monocytes or when NR4A1–/– donor lungs were used. Reconstitution of B6 NR4A1–/– lungs with WT B6 nonclassical monocytes prior to transplantation restored the levels of classical monocytes recruited to the donor allograft following reperfusion. Data are expressed as mean ± SEM. n = 5–8 per group. ****P < 0.001, 1-way ANOVA with post hoc Holm-Šídák test (E and F).

Figure 4. Spleen-derived classical monocytes promote neutrophil extravasation into lung grafts after transplantation.

(A) Arterial blood oxygenation was assessed, and absolute numbers of neutrophils were determined in (B) lung grafts 24 hours after transplantation of B6 CD45.1+ lungs into control or splenectomized congenic B6 CD45.2+ recipients. (C) Contour plots depicting gating strategy to identify intravascular vs. extravascular recipient neutrophils (CD45.2+CD11b+Gr-1+) and quantification of the percentage of graft-infiltrating neutrophils 24 hours after transplantation of B6 CD45.1+ lungs into control or splenectomized congenic B6 CD45.2+ hosts. Debris, dead cells, and doublets have been excluded from analysis based on forward scatter/side scatter (FSC/SSC) and FSC–pulse area [FSC-A]/FSC–pulse width [FSC-W] characteristics. (D) Absolute numbers of neutrophils were determined in the BALF 24 hours after transplantation of B6 CD45.1+ lungs into control or splenectomized congenic B6 CD45.2+ recipients. Data for A–D are expressed as median with interquartile range. n = 4 per group for arterial blood gas measurement; n = 7 per group for enumeration of neutrophils in lung grafts and BALF. *P < 0.05; **P < 0.01, Mann-Whitney U test. (E) Expression levels of Il1b, Tnfa, Cxcl1, Cxcl2, and Cxcl5 in B6 CD45.1+ lung grafts 24 hours after transplantation into control or splenectomized congenic B6 CD45.2+ hosts, assessed by quantitative real-time PCR. Results were normalized to Rn18s. Data are expressed as median with interquartile range. n = 5 per group. *P < 0.05; **P < 0.01, Mann-Whitney U test.

Figure 5. IL-1β production by graft-infiltrating monocytes is essential for neutrophil extravasation after lung transplantation.

(A) Recipient monocytes (CD45.2+CD11b+Ly6Chi) were isolated from the bone marrow or lung grafts in resting mice or 2 hours after transplantation of B6 CD45.1+ lungs into B6 CD45.2+ recipients, and their expression levels of Il1b, Tnfa, Cxcl1, Cxcl2, and Cxcl5 were analyzed by quantitative PCR. n = 4 each compartment with 1 statistical outlier for Cxcl1 and 1 statistical outlier for Cxcl5 in the lung excluded from analysis. *P < 0.05, Mann-Whitney U test. (B) Expression levels of IL1B in human donor grafts at conclusion of cold ischemia and 2 hours after reperfusion, assessed by quantitative PCR. n = 5. **P < 0.01, paired t test. (C) Arterial blood oxygenation was assessed and absolute numbers of recipient (D) monocytes and (E) neutrophils were determined in lung grafts 24 hours after transplantation of B6 CD45.1+ lungs into splenectomized congenic B6 CD45.2+ recipients that received either B6 WT or B6 IL-1β–deficient monocytes. Data are expressed as median with interquartile range. n = 6 per group. **P < 0.01, Mann-Whitney U test. (F) Representative contour plots of extravascular vs. intravascular neutrophils and quantification of the percentage of graft-infiltrating neutrophils in lung grafts and (G) number of neutrophils in BALF 24 hours after transplantation of B6 CD45.1+ lungs into splenectomized B6 CD45.2+ recipients that received B6 WT or B6 IL-1β–deficient monocytes. Data are expressed as median with interquartile range. n = 6 per group. *P < 0.05; **P < 0.01, Mann-Whitney U test. (H) Representative intravital 2-photon microscopy of B6 WT or B6 IL-1R–KO lung grafts 2 hours after transplantation into syngeneic B6 LysM-GFP mice and quantification of neutrophil extravasation. Qdot 655 labeled the vessels red. Recipient neutrophils are green. Scale bar: 50 μm. Data are expressed as median with interquartile range. n = 4 per group. *P < 0.05, Mann-Whitney U test.

Figure 6. MyD88 expression by graft-infiltrating monocytes is essential for neutrophil extravasation after lung transplantation.

(A) Expression levels of IL-1β were assessed by quantitative real-time PCR in bone marrow–derived B6 WT or MyD88-deficient monocytes after treatment with B6 lung lysates. Data are expressed as mean ± SEM of 3 biological replicates. **P < 0.01, unpaired t test. (B) Arterial blood oxygenation was assessed and absolute numbers of recipient (C) monocytes and (D) neutrophils were determined in lung grafts 24 hours after transplantation of B6 CD45.1+ lungs into splenectomized congenic B6 CD45.2+ recipients that received either B6 WT or B6 MyD88-deficient monocytes. (E) Representative contour plots of extravascular vs. intravascular neutrophils and quantification of the percentages of graft-infiltrated neutrophils in lung grafts as well as (F) number of neutrophils in BALF 24 hours after transplantation of B6 CD45.1+ lungs into splenectomized congenic B6 CD45.2+ recipients that received either B6 WT or B6 MyD88-deficient monocytes. Data are expressed as median with interquartile range. n = 5 per group. *P < 0.05; **P < 0.01, Mann-Whitey U test (B–F).

Figure 7. IL-1β mediates downregulation of ZO-2 in vascular endothelial cells and increases endothelial permeability.

Permeability was assessed in MLVEC cultures treated with (A) IL-1β at indicated concentrations and (B) after adding IL-1R–neutralizing (2.5 μg/ml) or control antibody for 24 hours. For A, a representative experiment of 3 biological replicates is shown. Data represent mean ± SD of 3 technical replicates. For B, data are presented as mean ± SD of 4 biological replicates. One statistical outlier was excluded in the group receiving IL-1β treatment only. **P < 0.01; ***P < 0.001; ****P < 0.001, 1-way ANOVA with post hoc Holm-Šídák test (A and B). RFU, relative fluorescence unit. (C) MLVECs were treated with IL-1β (0, 0.5, and 1 ng/ml) for 24 hours, fixed, and stained with ZO-2. Nuclei were counterstained with DAPI. Images are representative of 3 independent experiments. (D) MLVECs were treated with IL-1R–neutralizing or control antibody for 30 minutes prior to the addition of IL-1β (1 ng/ml). Cell lysates were analyzed for ZO-2 by Western blotting. The same blot was reprobed for GAPDH. Blots are representative of 3 independent experiments. (E) Frozen tissues of B6 CD45.1+ lung grafts, collected 24 hours after transplantation into splenectomized B6 CD45.2+ hosts that received B6 WT or B6 IL-1β–deficient monocytes and (F) human donor grafts at the conclusion of cold ischemia and 2 hours after reperfusion were stained with antibodies for ZO-2 (red) and CD31 (green). Nuclei were counterstained with DAPI. Arrowhead indicates colocalization of CD31 and ZO-2. Scale bar: 20 μm. Images were taken with a ×40 objective lens. The relative mean fluorescence intensity (MFI) of ZO-2 staining was quantified and is represented graphically for E and F. For E, data are expressed as median with interquartile range. n = 5 per group. *P < 0.05, Mann-Whitney U test. For F, n = 8 patients per group. *P < 0.05, paired t test.

Figure 8. Knockdown of ZO-2 increases endothelial permeability.

(A and B) Representative Western blot and semiquantitative analysis as well as (C) immunostaining of ZO-2 in MLVECs that were transfected with control siRNA or siRNA targeting ZO-2 for 48 hours. Scale bars: 20 μm. Blots and immunostaining are representative of 3 independent experiments each. Data are expressed as mean ± SD. n = 6 per group. ****P < 0.0001, 1-way ANOVA with post hoc Holm-Šídák test. (D) Endothelial permeability was assessed in MLVECs that were treated with control siRNA or siRNA targeting ZO-2, as described in the legend for Figure 7A. Data represent mean ± SD of 4 biological replicates. **P < 0.001, unpaired t test. (E) MLVECs were transfected with control siRNA or siRNA targeting ZO-2. Two days later, primary mouse bone marrow–derived neutrophils (1 x 105 cells) were added to the inserts. Twenty-four hours later, transmigrated neutrophils were enumerated in the lower chamber. Data represent mean ± SD of 3 biological replicates. *P < 0.05, unpaired t test.

Figure 9. Diagram depicting how spleen-derived classical monocytes mediate extravasation of neutrophils in reperfused lungs via MyD88/IL-1β–dependent pathway.

Classical monocytes are released from the spleen after reperfusion of lungs. After recruitment to the lung, they produce IL-1β in MyD88-dependent fashion. IL-1β signaling in endothelial cells results in the downregulation of ZO-2, which enhances endothelial permeability and facilitates neutrophil extravasation.