Heart-resident CCR2+ macrophages promote neutrophil extravasation through TLR9/MyD88/CXCL5 signaling

Abstract

It is well established that maladaptive innate immune responses to sterile tissue injury represent a fundamental mechanism of disease pathogenesis. In the context of cardiac ischemia reperfusion injury, neutrophils enter inflamed heart tissue, where they play an important role in potentiating tissue damage and contributing to contractile dysfunction. The precise mechanisms that govern how neutrophils are recruited to and enter the injured heart are incompletely understood. Using a model of cardiac transplant-mediated ischemia reperfusion injury and intravital 2-photon imaging of beating mouse hearts, we determined that tissue-resident CCR2⁺ monocyte-derived macrophages are essential mediators of neutrophil recruitment into ischemic myocardial tissue. Our studies revealed that neutrophil extravasation is mediated by a TLR9/MyD88/CXCL5 pathway. Intravital 2-photon imaging demonstrated that CXCL2 and CXCL5 play critical and nonredundant roles in guiding neutrophil adhesion and crawling, respectively. Together, these findings uncover a specific role for a tissue-resident monocyte-derived macrophage subset in sterile tissue inflammation and support the evolving concept that macrophage ontogeny is an important determinant of function. Furthermore, our results provide the framework for targeting of cell-specific signaling pathways in myocardial ischemia reperfusion injury.

Figure 1. Intravital 2-photon imaging reveals impaired neutrophil trafficking in heart grafts that lack monocyte-derived macrophages.

(A) Control PBS liposome–treated heart graft with neutrophil (green) arrest inside blood vessels (blood vessels appear red after injection of quantum dots), intravascular cluster formation, and extravasation (see Supplemental Video 1; n = 4 mice). Neutrophil trafficking in hearts derived from (B) donors that received treatment with clodronate liposomes prior to organ harvest (see Supplemental Video 2; n = 4 mice). Relative time is displayed in hrs:min:sec. Scale bars: 50 μm. (C) Percentage of neutrophils that extravasated during imaging period was significantly higher in hearts derived from control PBS liposome–treated donors compared with heart grafts harvested from clodronate liposome–treated mice. (D) Neutrophil rolling velocities were comparable in coronary veins of cardiac grafts derived from control PBS liposome–treated and clodronate liposome–treated mice. (E) Intraluminal crawling velocities were significantly lower in hearts harvested from clodronate liposome–treated WT compared with PBS liposome–treated mice. *P < 0.05; **P < 0.01 (t test). Data in C, D, and E are derived from 4 mice for each experimental group. For D and E, symbols represent averages obtained from individual mice with over 30 neutrophils examined per mouse, horizontal bars denote means, and error bars denote ±SEM.

Figure 2. Intravital 2-photon imaging reveals impaired neutrophil trafficking in heart grafts that lack CCR2⁺ monocytes and monocyte-derived macrophages.

(A) Dot plots depict flow cytometric analysis of macrophage/monocyte populations in B6 CD45.2⁺ heart grafts 2 hours after transplantation into congenic B6 CD45.1⁺ hosts. CCR2^–MHCII^hi, CCR2^–MHCII^lo, and CCR2⁺MHCII^hi macrophages and CCR2⁺MHCII^lo monocytes are present. Macrophage/monocyte populations are gated on donor hematopoietic (CD45.2⁺CD45.1^–) and myeloid (CD11b⁺CD64⁺) cells. Plots are representative of 4 independent experiments with comparable results. (B) Quantification of donor monocyte/macrophage populations represented as percentage of CD45.2⁺CD11b⁺CD64⁺ cells based on gating depicted in A. Neutrophil (green) trafficking in (C) control diphtheria toxin–treated (DT-treated) WT (see Supplemental Video 3; n = 4 mice)or (D) DT-treated CCR2-DTR (DT receptor) heart grafts (see Supplemental Video 4; n = 5 mice). Blood vessels appear red after injection of quantum dots (n = 4 mice). (E) Percentage of neutrophils that extravasated during imaging period was significantly higher in hearts derived from DT-treated WT donors compared with heart grafts harvested from DT-treated CCR2-DTR mice. (F) Neutrophil rolling velocities were comparable in coronary veins of cardiac grafts derived from DT-treated WT and DT-treated CCR2-DTR mice. (G) Intraluminal crawling velocities were significantly lower in hearts harvested from DT-treated CCR2-DTR compared with DT-treated WT mice. **P < 0.01 (t test). Data in E, F, and G are derived from 4 mice receiving hearts from DT-treated WT mice and 5 recipients of cardiac grafts derived from DT-treated CCR2-DTR donors. For F and G, symbols represent averages obtained from individual mice with over 30 neutrophils examined per mouse, horizontal bars denote means, and error bars denote ±SEM.

Figure 3. Intravital 2-photon imaging reveals impaired neutrophil extravasation into heart grafts that lack MyD88 expression.

Neutrophil trafficking in (A) WT (see Supplemental Video 5; n = 4 mice) and (B) MyD88-deficient hearts (see Supplemental Video 6; n = 4 mice). Scale bars: 50 μm. (C) Percentage of neutrophils that entered myocardial tissue during imaging period was significantly lower when heart grafts were deficient in MyD88 compared with WT cardiac grafts. (D) Neutrophil rolling velocities were significantly higher in coronary veins of cardiac grafts that lacked expression of MyD88 when compared with WT hearts. (E) Intraluminal crawling velocities of neutrophils were significantly lower when hearts lacked expression of MyD88 when compared with WT cardiac grafts. Neutrophil trafficking in (F) cardiac grafts derived from control MyD88-floxed mice (see Supplemental Video 8; n = 4 mice) and (G) mice that lack MyD88 selectively in Lysozyme M⁺ cells (see Supplemental Video 9; n = 4 mice). Relative time is displayed in hrs:min:sec. Scale bars: 50 μm. (H) Percentage of neutrophils that entered myocardial tissue during imaging period was significantly lower when heart grafts were deficient in MyD88 in Lysozyme M–expressing cells compared with control MyD88-floxed hearts. (I) Neutrophil rolling velocities were significantly higher in coronary veins of cardiac grafts that lacked expression of MyD88 in Lysozyme M–expressing cells compared with control MyD88-floxed hearts. (J) Intraluminal crawling velocities of neutrophils were significantly lower when hearts lacked expression of MyD88 in Lysozyme M–expressing cells compared with control MyD88-floxed hearts. *P < 0.05; **P < 0.01; ***P < 0.001 (t test). Data in C–E and H–J are derived from 4 mice for each experimental group. C–E and H–J represent averages obtained from individual mice with over 30 neutrophils examined per mouse, horizontal bars denote means, and error bars denote ±SEM.

Figure 4. Expression levels of neutrophil chemoattractants are decreased in donor macrophage populations in MyD88-deficient heart grafts.

Expression levels of CXCL2 and CXCL5 in sorted donor-derived (CD45.2⁺CD45.1^–CD11b⁺CD64⁺) CCR2⁺ and CCR2^– macrophages examined 2 hours after transplantation of B6 CD45.2⁺ WT or B6 CD45.2⁺ MyD88–deficient hearts into congenic B6 CD45.1⁺ recipients. Results were normalized to 18s RNA and compared with the CCR2^– macrophage population in each experimental group. *P < 0.05; **P < 0.01 (2-way ANOVA). Filled circles, WT CCR2^- macrophages; filled squares, WT CCR2⁺ macrophages and monocyte-derived macrophages; filled triangles, MyD88-deficient CCR2^– macrophages; open circles, MyD88-deficient CCR2⁺ macrophages and monocyte-derived macrophages. Graph represents at least 4 separate experiments per group where cells from 2 hearts were pooled for each experiment. Horizontal bars denote means, and error bars denote ±SEM.

Figure 5. Intravital 2-photon imaging demonstrates impaired neutrophil extravasation into TLR9-deficient heart grafts.

Neutrophil trafficking in (A) TLR2-deficient (see Supplemental Video 10; n = 4 mice), (B) toll-interleukin 1 receptor domain containing adaptor protein–deficient (TIRAP-deficient; see Supplemental Video 11; n = 4 mice), and (C) TLR9-deficient (see Supplemental Video 12; n = 4 mice) cardiac grafts. White arrows in A and B point to sites of neutrophil extravasation. Relative time is displayed in hrs:min:sec. Scale bars: 50 μm. (D) Percentage of neutrophils that entered myocardial tissue during imaging period was comparable between WT, TLR2-deficient, and TIRAP-deficient hearts. However, the percentage of extravasated neutrophils was significantly lower in TLR9-deficient than WT cardiac grafts. (E) Neutrophil rolling velocities were significantly higher in coronary veins of TLR9-deficient cardiac grafts when compared with WT hearts. Rolling velocities in TLR2- or TIRAP-deficient hearts were comparable with WT cardiac grafts. (F) Intraluminal crawling velocities of neutrophils did not differ significantly between WT, TLR2-deficient, and TIRAP-deficient hearts but were significantly lower than WT conditions when hearts lacked expression of TLR9. *P < 0.05; **P < 0.01 (one-way ANOVA). Data in D, E, and F are derived from 4 mice for each experimental group. For D, E, and F, symbols represent averages obtained from individual mice with over 30 neutrophils examined per mouse, horizontal bars denote means, and error bars denote ±SEM.

Figure 6. Expression levels of neutrophil chemoattractants are decreased in donor macrophage populations in TLR9-deficient heart grafts.

Expression levels of CXCL2 and CXCL5 in sorted donor-derived (CD45.2⁺CD45.1^–CD11b⁺CD64⁺) CCR2⁺ and CCR2^– macrophages examined 2 hours after transplantation of B6 CD45.2⁺ WT or B6 CD45.2⁺ MyD88–deficient hearts into congenic B6 CD45.1⁺ recipients. Results were normalized to 18s RNA and compared with the CCR2^– macrophage population in each experimental group. *P < 0.05; **P < 0.01 (2-way ANOVA). Filled circles, WT CCR2^- macrophages; filled squares, WT CCR2⁺ macrophages and monocyte-derived macrophages; filled triangles, TLR9-deficient CCR2^– macrophages; open circles, TLR9-deficient CCR2⁺ macrophages and monocyte-derived macrophages. Graph represents at least 4 separate experiments per group where cells from 2 hearts were pooled for each experiment. Horizontal bars denote means, and error bars denote ±SEM.

Figure 7. CXCL2 and CXCL5 regulate neutrophil chemotaxis and trafficking of neutrophils in heart grafts.

Neutrophil trafficking in WT hearts after administration of (A) isotype control antibodies (see Supplemental Video 13; n = 4 mice) or (B) CXCL2-neutralizing antibody (see Supplemental Video 14; n = 4 mice). Relative time is displayed in hrs:min:sec. Scale bars: 50 μm. (C) Percentage of neutrophils that entered myocardial tissue during imaging period was significantly lower after neutralization of CXCL2 compared with mice that received isotype control antibodies. (D) Neutrophil rolling velocities were significantly higher in coronary veins of cardiac grafts after neutralization of CXCL2 compared with mice that received isotype control antibodies. (E) Due to a small number of adherent neutrophils, crawling velocities could not be evaluated when CXCL2 was neutralized. Neutrophil trafficking in (F) WT and (G) CXCL5-deficient cardiac grafts (see Supplemental Videos 5 and 15, respectively; Isotype control, n = 4; CXCL2-neutralizing antibody, n = 4; CXCL5-deficient heart grafts, n = 5. Relative time is displayed in hrs:min:sec. Scale bars: 50 μm. (H) Percentage of neutrophils that entered myocardial tissue during imaging period was significantly lower when cells in the hearts were unable to produce CXCL5 compared with WT cardiac grafts. (I) Neutrophil rolling velocities were significantly higher in coronary veins of cardiac grafts when hearts lacked expression of CXCL5 compared with WT cardiac grafts. (J) Intraluminal crawling velocities of neutrophils were significantly lower when hearts lacked expression of CXCL5 compared with WT cardiac grafts. *P < 0.05; **P < 0.01; ***P < 0.001 (t test). Data in C–E and H–J are derived from 4–5 mice for each experimental group as indicated above. For C–E and H–J, symbols represent averages obtained from individual mice with over 30 neutrophils examined per mouse, horizontal bars denote means, and error bars denote ±SEM.

Figure 8. Model of CCR2⁺ macrophage–mediated neutrophil extravasation.

Tissue-resident CCR2⁺ macrophages regulate neutrophil trafficking in injured hearts through TLR9/MyD88 pathway–dependent production of CXCL2 and CXCL5.