PMN-derived netrin-1 attenuates cardiac ischemia-reperfusion injury via myeloid ADORA2B signaling

Abstract

Previous studies implicated the neuronal guidance molecule netrin-1 in attenuating myocardial ischemia-reperfusion injury. However, the tissue-specific sources and receptor signaling events remain elusive. Neutrophils are among the first cells responding to an ischemic insult and can be associated with tissue injury or rescue. We found netrin-1 levels were elevated in the blood of patients with myocardial infarction, as well as in mice exposed to myocardial ischemia-reperfusion. Selectively increased infarct sizes and troponin levels were found in Ntn1loxP/loxP Lyz2 Cre+ mice, but not in mice with conditional netrin-1 deletion in other tissue compartments. In vivo studies using neutrophil depletion identified neutrophils as the main source for elevated blood netrin-1 during myocardial injury. Finally, pharmacologic studies using treatment with recombinant netrin-1 revealed a functional role for purinergic signaling events through the myeloid adenosine A2b receptor in mediating netrin-1-elicited cardioprotection. These findings suggest an autocrine signaling loop with a functional role for neutrophil-derived netrin-1 in attenuating myocardial ischemia-reperfusion injury through myeloid adenosine A2b signaling.

Figure 1.

Blood netrin-1 increases in MI patients and mice with myocardial IR surgery. (A) Blood netrin-1 levels of healthy donors and MI patients (n = 10 for healthy control, n = 9 for MI; two-tailed Welch’s t test). (B) Experimental strategy for the murine myocardial IR model. (C) Infarct sizes of IR group compared with the sham group (n = 4 per group; two-tailed unpaired t test). (D) Representative infarct staining results for the IR model. The infarct area is outlined by a green line; the blue line marks the AAR (scale bar = 1 mm). (E) cTnI levels after surgery (n = 6 for sham group, n = 9 for IR group; two-tailed Welch’s t test). (F) Murine blood netrin-1 levels were measured in sham, ischemia (45 min), IR2h (45 min of ischemia + 2 h of reperfusion), and IR4h (45 min of ischemia + 4 h of reperfusion) groups (n = 6 for sham group, n = 5 for ischemia group, n = 9 for IR2h group, and n = 5 for IR4h group; one-way ANOVA with Bonferroni post hoc tests). (G) Immunofluorescence costaining of netrin-1 with cTnT and Ly6G in WT mice after sham or IR surgery shows netrin-1 in cardiomyocytes and neutrophils. (For single-channel pictures, scale bar = 2 µm; for merged channel pictures, scale bar = 8 µm.) *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Data are presented as mean ± SD.

Figure S1.

Netrin-1 levels showed no differences in murine IR heart tissue and blood of female/male subgroups. (A and B) Murine heart tissue netrin-1 levels were measured in sham and IR2h groups with Western blot analysis. Quantifications showed netrin-1 levels in heart tissue did not change significantly after 45 min of ischemia and 2 h of reperfusion (n = 5 for each group; two-tailed unpaired t test). (C and D) Murine IR injury model shows no significant differences of blood netrin-1 levels between female and male mice in both sham and IR groups (n = 3 per sham group, n = 5 for female IR group, and n = 4 for male IR group; two-tailed unpaired t test). Data are presented as mean ± SD.

Figure S2.

Netrin-1 staining of heart tissue, negative controls of immunofluorecence, and blood netrin-1 levels of monocyte/macrophage-depleted mice. (A) Immunohistochemical staining for cardiac netrin-1 in WT mice after IR surgery shows positive staining in cardiomyocytes and neutrophils (n = 3; *, cardiomyocytes; #, neutrophils; for original pictures, scale bar = 131.8 µm; for enlarged pictures, scale bar = 41.7 µm). (B) Negative controls for Fig. 1 G (for single-channel pictures, scale bar = 2 µm; for merged channel pictures, scale bar = 8 µm). (C) Negative controls for Fig. 3 H. Scale bar = 10 µm. (D) Murine blood monocyte cell counts after clodronate depletion compared with liposome control group (n = 3 for each group; two-tailed unpaired t test). (E) Blood netrin-1 levels of WT (C57BL/6) mice with clodronate monocyte/macrophage depletion after IR surgery compared with sham group (n = 5 for each group; Mann-Whitney test). *, P < 0.05; **, P < 0.01. Data are presented as mean ± SD.

Figure S3.

Confirmations of tissue-specific netrin-1 knock-down mice. (A) Representative Western blot shows the confirmation of netrin-1 knock-down in heart tissue of Ntn1^loxP/loxP Myosin Cre⁺ mice (n = 4). (B) Netrin-1 transcription levels of bone marrow cells determined by RT-qPCR show netrin-1 knock-down in the myeloid compartment of Ntn1^loxP/loxP Lyz2 Cre⁺ mice (n = 3; two-tailed Welch’s t test; *, P < 0.05. Data are presented as mean ± SD).

Figure 2.

Ntn^loxP/loxP Lyz2 Cre⁺ mice have severe injury after myocardial IR surgery. (A) Experimental strategy for myocardial IR model. (B) Infarct sizes of Myosin Cre⁺ mice compared with Ntn1^loxP/loxP Myosin Cre⁺ mice after IR surgery (n = 6 for Myosin Cre⁺, n = 5 for Ntn1^loxP/loxP Myosin Cre⁺; two-tailed unpaired t test). (C) Representative infarct staining results of Myosin Cre⁺ mice and Ntn1^loxP/loxP Myosin Cre⁺ mice. The infarct area is outlined by a green line, and the blue line marks the AAR (scale bar = 1 mm). (D) cTnI levels of Myosin Cre⁺ mice compared with Ntn1^loxP/loxP Myosin Cre⁺ mice after surgery (n = 6 for Myosin Cre⁺, n = 5 for Ntn1^loxP/loxP Myosin Cre⁺; Mann-Whitney test). (E) Infarct sizes of Lyz2 Cre⁺ mice compared with Ntn1^loxP/loxP Lyz2 Cre⁺ mice after IR surgery (n = 5 for Lyz2 Cre⁺, n = 7 for Ntn1^loxP/loxP Lyz2 Cre⁺; two-tailed unpaired t test). (F) Representative infarct staining results of Lyz2 Cre⁺ mice and Ntn1^loxP/loxP Lyz2 Cre⁺ mice as described in C (scale bar = 1 mm). (G) cTnI levels of Lyz2 Cre⁺ mice compared with Ntn1^loxP/loxP Lyz2 Cre⁺ mice after surgery (n = 5 for Lyz2 Cre⁺, n = 7 for Ntn1^loxP/loxP Lyz2 Cre⁺; two-tailed unpaired t test). *, P < 0.05; **, P < 0.01. Data are presented as mean ± SD.

Figure 3.

Neutrophils release netrin-1 in response to murine cardiac IR injury and in vitro activation. (A and B) Surgery strategy and blood netrin-1 levels of Lyz2 Cre⁺ mice compared with Ntn1^loxP/loxP Lyz2 Cre⁺ mice after IR surgery (n = 5 for Lyz2 Cre⁺, n = 5 and 7 for Ntn1^loxP/loxP Lyz2 Cre⁺ sham and IR groups, respectively; two-way ANOVA with Bonferroni post hoc tests). (C) Neutrophil depletion and IR surgery strategy (IgG, IgG isotype control group; αLy6G, neutrophil depletion group). (D) Murine blood neutrophil cell counts after neutrophil depletion (n = 5 for isotype IgG control, n = 4 for αLy6G neutrophil depletion group; Mann-Whitney test). (E) Blood netrin-1 levels of WT (C57BL/6) mice with neutrophil depletion or IgG control after IR surgery (n = 7 for isotype IgG control, n = 5 for αLy6G neutrophil depletion group; two-way ANOVA with Bonferroni post hoc tests). (F and G) Netrin-1 level changes in supernatants of human PMN (hPMN) after being activated with TNFα or fMLP for 2 h; PBS or DMSO was used as a control, respectively (TNFα and PBS groups, data from three independent experiments; fMLP and DMSO groups, data from four independent experiments; two-tailed unpaired t test). (H) Immunofluorescence costaining of netrin-1 with lysozyme in human PMN shows that netrin-1 locates in the cytosol but not in the neutrophil lysozyme-containing granules. (For left four pictures, scale bar = 8 µm; for enlarged three pictures on the right, scale bar = 2 µm.) *, P < 0.05; **, P < 0.01. Data are presented as mean ± SD.

Figure 4.

Cardioprotection of netrin-1 is ADORA2B dependent. (A) Surgery and netrin-1 treatment strategy. Recombinant mouse netrin-1 or PBS was given i.v. 5 min before reperfusion. (B) Infarct sizes of Adora2b^−/− mice compared with WT mice after IR surgery with NTN1 treatment or PBS control (n = 10 for Adora2b^−/− groups, n = 5 for WT groups; two-way ANOVA with Bonferroni post hoc tests). (C) Representative infarct staining results of Adora2b^−/− mice and WT mice. The infarct area is outlined by a green line, and the blue line marks the AAR (scale bar = 1 mm). (D) cTnI levels of Adora2b^−/− mice compared with WT mice after surgery and netrin-1 treatment (n = 10 for Adora2b^−/− group, n = 7 for WT group; two-way ANOVA with Bonferroni post hoc tests). (E) ADORA2B receptor antagonist PSB1115 and netrin-1 treatment strategy. Recombinant mouse netrin-1 or PBS was given i.v. 5 min before reperfusion. (F) Infarct sizes of PSB1115-treated mice compared with vehicle control group after IR surgery with or without netrin-1 treatment (n = 10 for PSB1115-treated group, n = 5 and 6 for vehicle control group; two-way ANOVA with Bonferroni post hoc tests). (G) Representative infarct staining results of PSB1115-treated group and vehicle group. The infarct area is outlined by a green line, and the blue line marks the AAR (scale bar = 1 mm). (H) cTnI levels of PSB1115-treated mice compared with vehicle control mice after surgery and netrin-1 treatment (n = 10 for PSB1115-treated group, n = 7 and 8 for vehicle control group; two-way ANOVA with Bonferroni post hoc tests). *, P < 0.05. Data are presented as mean ± SD.

Figure S4.

Therapeutic effects of netrin-1 given in different dosages or time points and adenosine levels changes after netrin-1 treatment. (A) Recombinant mouse netrin-1 (NTN1) was given to WT mice 5 min before reperfusion during IR surgery at different dosages (5 µg/kg, 10 µg/kg, and 20 µg/kg, equal volumes of PBS as control). Infarct sizes showed that 20 µg/kg netrin-1 presented significant cardioprotection (n = 5 for PBS group, n = 4 for 5 µg/kg group, n = 5 for 10 µg/kg group, and n = 4 for 20 µg/kg group; one-way ANOVA with Bonferroni post hoc tests). (B and C) Recombinant mouse netrin-1 (NTN1) was given to WT mice 5 min, 25 min, or 45 min before reperfusion during IR surgery (20 µg/kg or equal volume of PBS). Infarct sizes (B) and troponin levels (C) showed protective effects from three different treatment time points. (D and E) Mouse serum adenosine levels and heart tissue adenosine levels showed no significant changes after netrin-1 treatment (20 µg/kg netrin-1; adenosine levels were measured at 5 min, 25 min, and 45 min after netrin-1 injection via HPLC). *, P < 0.05; **, P < 0.01. Data are presented as mean ± SD.

Figure 5.

Cardioprotection of netrin-1 is myeloid ADORA2B dependent. (A) Surgery and netrin-1 treatment strategy. Recombinant mouse netrin-1 or PBS was given i.v. 5 min before reperfusion. (B) Infarct sizes of Adora2b^loxP/loxP VE-cadherin Cre⁺ mice compared with VE-cadherin Cre⁺ mice (n = 6 per group; two-tailed unpaired t test). (C, F, and I) Representative infarct staining results of Adora2b^loxP/loxP Cre⁺ and corresponding Cre⁺ mice. The infarct area is outlined by a green line, and the blue line marks the AAR (scale bar = 1 mm). (D) cTnI levels of Adora2b^loxP/loxP VE-cadherin Cre⁺ mice compared with VE-cadherin Cre⁺ (n = 6 and 5 for PBS and netrin-1 treatment groups, respectively; two-tailed unpaired t test). (E) Infarct sizes of Adora2b^loxP/loxP Myosin Cre⁺ mice compared with Myosin Cre⁺ mice (n = 6 per group; two-tailed unpaired t test). (G) cTnI levels of Adora2b^loxP/loxP Myosin Cre⁺ mice compared with Myosin Cre⁺ (n = 6 per group; two-tailed Welch’s t test). (H) Infarct sizes of Adora2b^loxP/loxP Lyz2 Cre⁺ mice compared with Lyz2 Cre⁺ mice (n = 7 and 8 for PBS and netrin-1 treatment groups, respectively; two-tailed unpaired t test). (J) cTnI levels of Adora2b^loxP/loxP Lyz2 Cre⁺ mice compared with Lyz2 Cre⁺ (n = 7 and 8 for PBS and netrin-1 treatment groups, respectively; two-tailed unpaired t test). ****, P < 0.0001; *, P < 0.05. Data are presented as mean ± SD.

Figure S5.

Netrin-1 reduces neutrophil transmigration, which is ADORA2B dependent. (A) Murine neutrophil transmigration assay results show NTN1 reduces murine neutrophil transmigration and ADORA2B receptor antagonist PSB1115 abolishes the effect (n = 4 per group; one-way ANOVA with Bonferroni post hoc tests). Data from four independent experiments. (B and C) Human neutrophil transmigration assay 1-h (B) and 2-h (C) results show NTN1 reduces human neutrophil transmigration and ADORA2B receptor antagonist PSB1115 abolishes the effect (n = 4 per group; one-way ANOVA with Bonferroni post hoc tests). **, P < 0.01; ***, P < 0.001. Data are presented as mean ± SD. Data from four independent experiments.

Figure 6.

Netrin-1 reduced neutrophil infiltration after cardiac IR is myeloid ADORA2B dependent. (A) Surgery and netrin-1 treatment strategy. Recombinant mouse netrin-1 or PBS was given i.v. 5 min before reperfusion. (B and C) Neutrophil infiltration (CD45⁺Ly6G⁺) in Lyz2 Cre⁺ mouse hearts after surgery was determined by flow cytometry (n = 4, 4, and 3 for sham group, PBS group, and NTN1 group, respectively; one-way ANOVA with Bonferroni post hoc tests). (D and E) Neutrophil infiltration (CD45⁺Ly6G⁺) in Adora2b^loxP/loxP Lyz2 Cre⁺ mouse hearts after surgery determined by flow cytometry (n = 3, 4, and 4 for sham group, PBS group, and netrin-1 group, respectively; one-way ANOVA with Bonferroni post hoc tests). **, P < 0.01; ***, P < 0.001. Data are presented as mean ± SD.