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. 2022 Apr 11;26(1):14.
doi: 10.1186/s40824-022-00260-y.

M2-like macrophages transplantation protects against the doxorubicin-induced heart failure via mitochondrial transfer

Yihai Liu  1   2 , Mingyue Wu  1 , Chongxia Zhong  1 , Biao Xu  3 , Lina Kang  4
Affiliations

M2-like macrophages transplantation protects against the doxorubicin-induced heart failure via mitochondrial transfer

Yihai Liu et al. Biomater Res. .

Abstract

Aims: The alternatively activated macrophages have shown a cardioprotective effect in heart failure. However, the effect of M2 adoptive transfer in non-ischemic heart failure is unknown. In this study, we evaluated the efficacy of M-CSF plus IL-4 induced M2-like macrophages transplantation in doxorubicin-induced cardiotoxicity.

Methods: Bone marrow mononuclear cells were polarized as CCR2+CD206+ M2-like macrophages by a combination of M-CSF plus IL-4 treatment. C57BL/6 mice received a single intraperitoneal injection of doxorubicin (15 mg/kg). The treatment group were treated with M2-like macrophages (1 × 10^6 cells per mouse; i.v.) once a week for 2 weeks. After 3 weeks, we examined the percentage of resident cells and cardiac function. Furthermore, we evaluated cardiac fibrosis, cardiomyocyte apoptosis and circulating inflammatory factors. Finally, we investigated the mitochondria transfer in vitro in a direct and indirect co-culture conditions.

Results: Cardiac function was significantly improved in doxorubicin-induced heart failure by adoptive transfer of M2-like macrophages. Besides, M2-like macrophages treatment attenuated cardiac fibrosis and cardiomyocyte apoptosis, as well as increased the level of circulating IL-4 and Th2 response. In vitro, M2-like macrophages could transfer mitochondria to injured cardiomyocytes in a direct and indirect way.

Conclusions: In our study, adoptive transfer of M2-like macrophages could protect against the doxorubicin-induced cardiotoxicity, which may be partly attributed to mitochondria transfer. And M2-like macrophages transplantation could become a treatment for non-ischemic heart failure in the clinical practice.

Keywords: Cardiotoxicity; Cell transplantation; Heart failure; M2-like macrophages; Mitochondria.

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Conflict of interest statement

There is no competing interest to declare.

Figures

Fig. 1
Fig. 1
Production of M2-like macrophages by M-CSF + IL-4 treatment. A The cell morphology was shown under the light microscopy. M0 macrophages (without M-CSF plus IL-4 treatment) were used as controls. Scale bar 100 μm. B The surface expression of CCR2 and CD206 was measured using flow cytometry. Below was the fluorescent intensity of CCR2 and CD206, and the quantification results. C The gene expression of M1- and M2-related markers, including IL-1β, TNF-α, iNOS and IL-10, TGF-β, Arg-1, were measured by qPCR. (Data were presented as mean ± SD. ***p < 0.001. N = 3 per group)
Fig. 2
Fig. 2
Cardiac function between groups were measured using transthoracic echocardiography between groups. A Representative echocardiography images between groups. B Ejection fraction (EF), C Fractional shortening (FS) and D left ventricular internal dimension (LVID) at systole were measure among groups. (Data were presented as mean ± SD. ***P < 0.001 vs. Sham; #P < 0.05 vs. DOX. N = 5 per group, One-way ANOVA)
Fig. 3
Fig. 3
Histological staining was used to evaluate cardiac remodeling among groups. A HE staining for quantification of cardiomyocyte vacuoles. B Masson staining for quantification of interstitial fibrosis. C Wheat germ agglutinin staining for measurement of cell size. The arrowheads showed corresponding alterations. Three microscopic fields were analyzed per animal. (Data were presented as mean ± SD. ***P < 0.001 vs. Sham; ###P < 0.001 vs. DOX. N = 5 per group)
Fig. 4
Fig. 4
The circulating inflammatory factors were measured by enzyme-linked immunosorbent assay after 3 weeks. The levels of A IL-1β, B IL-4, C IL-6, and D IL-10 among groups. (Data were presented as mean ± SD. *P < 0.05 vs. Sham; ###P < 0.001 vs. DOX. N = 5 per group)
Fig. 5
Fig. 5
M2-like macrophages induced the Th2 response within myocardium. A The CD4+IL-4+Th2 cells were measured using flow cytometry and were quantified as the percentage of cardiac cells. B The presence of GATA3+ cells within the hearts were observed by immunofluorescence staining and were quantified as the percentage of cardiac cells. The arrowhead showed the GATA3+ cells. (Data were presented as mean ± SD. *P < 0.05 vs. Sham; #P < 0.05, ##P < 0.01 vs. DOX. N = 5 per group)
Fig. 6
Fig. 6
M2-like macrophages inhibited cardiomyocyte apoptosis. A The TUNEL staining of apoptotic cardiac cells. The arrowhead showed the TUNEL-positive cell. B The immunoblotting results among groups and the gray value of t-AKT, p-AKT, t-ERK 1/2, p-ERK 1/2, and c-caspase 3 from up-left to down-right. (Data were presented as mean ± SD. *P < 0.05, **P < 0.01 vs. Sham; #P < 0.05, ##P < 0.01 vs. DOX. N = 5 per group)
Fig. 7
Fig. 7
M2-like macrophages could transfer mitochondria to cardiomyocytes in vivo and in vitro. A The green fluorescence showed the presence of M2-like macrophages within the myocardium while the red positive mitochondria were localized inside the myocardium. The quantitative results were shown as the histogram at the left-bottom. B Red fluorescence dots (mitochondria) were incorporated into the body of CSFE-labelled cardiomyocytes. (Data were presented as mean ± SD. ***P < 0.001. N = 3 per group)
Fig. 8
Fig. 8
Transfer of Mitochondria from M2 macrophage to cardiomyocyte in a transwell plate (8 μm pore). A Up: M2 macrophage derived mitochondrial were located inside the body of cardiomyocytes. B Middle: M2 condition medium derived mitochondrial were internalized into the body of cardiomyocytes. C Down: No mitochondria were taken by cardiomyocytes using mitochondria-depleted condition medium of M2-like macrophages
Fig. 9
Fig. 9
Mitochondria internalization alleviated the cell stress induced by DOX. A Mitochondria treatment decreased the LDH release level. B Mitochondria treatment decreased the percentage of Annexin V + PI- and Annexin V + PI + cells. (Data were presented as mean ± SD. *P < 0.05, ***P < 0.001 vs. Sham; #P < 0.05 vs. DOX. N = 3 per group)
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