Modulating donor mitochondrial fusion/fission delivers immunoprotective effects in cardiac transplantation

Abstract

Early insults associated with cardiac transplantation increase the immunogenicity of donor microvascular endothelial cells (ECs), which interact with recipient alloreactive memory T cells and promote responses leading to allograft rejection. Thus, modulating EC immunogenicity could potentially alter T cell responses. Recent studies have shown modulating mitochondrial fusion/fission alters immune cell phenotype. Here, we assess whether modulating mitochondrial fusion/fission reduces EC immunogenicity and alters EC-T cell interactions. By knocking down DRP1, a mitochondrial fission protein, or by using the small molecules M1, a fusion promoter, and Mdivi1, a fission inhibitor, we demonstrate that promoting mitochondrial fusion reduced EC immunogenicity to allogeneic CD8⁺ T cells, shown by decreased T cell cytotoxic proteins, decreased EC VCAM-1, MHC-I expression, and increased PD-L1 expression. Co-cultured T cells also displayed decreased memory frequencies and Ki-67 proliferative index. For in vivo significance, we used a novel murine brain-dead donor transplant model. Balb/c hearts pretreated with M1/Mdivi1 after brain-death induction were heterotopically transplanted into C57BL/6 recipients. We demonstrate that, in line with our in vitro studies, M1/Mdivi1 pretreatment protected cardiac allografts from injury, decreased infiltrating T cell production of cytotoxic proteins, and prolonged allograft survival. Collectively, our data show promoting mitochondrial fusion in donor ECs mitigates recipient T cell responses and leads to significantly improved cardiac transplant survival.

Keywords: animal models: murine; basic (laboratory) research/science; heart transplantation/cardiology; immunobiology; immunosuppression/immune modulation; ischemia reperfusion injury (IRI); rejection: T cell mediated (TCMR); rejection: acute; translational research/science.

FIGURE 1

Genetic alteration of mitochondrial fission machinery reduces EC immunogenicity. (A) Representative 3D confocal images showing mitochondrial elongation in ECs that have dynamin‐related protein 1 (DRP1) knocked down (referred to as DRP1KDs) (red: mitochondria, blue: nuclei), and quantification of mitochondrial morphology in cells (hyperfused: at least one mitochondria ≥5 μm in length, intermediate: at least one mitochondria between 5 and 2 μm but none more than 5 μm, rounded: none longer than 2 μm) (n = 4 with 80 cells/group). (B) Mitochondrial mass of DRP1KD versus MCEC (the wildtype control) quantified by flow cytometry for mean fluorescence intensity of Mitotracker Green (n = 3). (C) Mitochondrial fitness of DRP1KD versus MCEC assessed by Seahorse flux assay (n = 4). (D) Immunogenicity of DRP1KD versus MCEC when being co‐cultured with allogeneic CD8⁺ T cells, demonstrated by supernatant levels of interferon gamma and granzyme‐B levels after 7 days of coculturing (n = 3). (E) Representative 3D confocal images showing mitochondrial elongation is unchanged at 1 h post‐reperfusion in DRP1KDs that were subjected to static cold storage for 6 h prior to reperfusion with warm media (red: mitochondria, blue: nuclei), and quantification of mitochondrial morphology performed as in Figure 1A (n = 4 with 80 cells/group). (F) Immunogenicity of DRP1KD versus MCEC that were subjected to cold storage for 6 h prior to reperfusion with warm media and co‐cultured with allogeneic CD8⁺ T cells, demonstrated by supernatant levels of interferon gamma and granzyme‐B after 7 days of coculturing (n = 3). (****p < .0001, ***p < .005, **p < .01, *p < .05. Differences between DRP1KD versus MCEC were analyzed using Student's t‐test. In panels D and F, granzyme‐B and interferon gamma levels were normalized to the number of ECs at the initiation of co‐culture to account for target differences)

FIGURE 2

Pharmacological modulation of mitochondrial fusion/fission prior to pro‐inflammatory cytokine insult reduces EC immunogenicity. (A) Representative 3D confocal images showing mitochondrial elongation in M1/Mdivi1‐treated MCECs (red: mitochondria, blue: nuclei), and quantification of mitochondrial morphology performed as in Figure 1A (n = 4 with 80 cells/group). (B) Immunogenicity of M1/Mdivi1‐pretreated cells versus untreated control co‐cultured with allogeneic CD8⁺ T cells, demonstrated by supernatant interferon gamma and granzyme‐B levels after 7 days of coculturing (n = 3). (C) VCAM‐1, ICAM‐1 adhesion molecules expression on the surface of MCECs pretreated with M1/Mdivi1 for 24 h versus untreated control prior to activation by TNFα/IFNγ (n = 4), and PD‐L1 expression on the surface of M1/Mdivi1‐pretreated MCECs after 4 days co‐cultured with CD8⁺ T cells (n = 3). (D) Abrogation of tolerogenic effect induced by M1/Mdivi1 when PD‐L1 is blocked, demonstrated by supernatant interferon gamma and granzyme‐B levels after 7 days of co‐culturing in the presence of anti‐PD‐L1 blocking antibodies (n = 3). (****p < .0001, ***p < .005, **p < .01, *p < .05. Differences between untreated control and M1/Mdivi1‐treated groups, as well as between PD‐L1‐unblocked control and PD‐L1‐blocked groups, were analyzed using Student's t‐test. In panels B and D, granzyme‐B and interferon gamma levels were normalized to the number of ECs at the initiation of co‐culture to account for target differences)

FIGURE 3

Pharmacological modulation of mitochondrial fusion/fission prior to cold storage reduces EC immunogenicity after reperfusion. (A) Representative 3D confocal images showing mitochondrial elongation is still retained at 1 h post‐reperfusion in MCECs pretreated with M1/Mdivi1 normothermically prior to 6‐h cold storage and warm reperfusion (red: mitochondria, blue: nuclei), and quantification of mitochondrial morphology performed as in Figure 1A (n = 4 with 80 cells/group). (B) Immunogenicity of MCECs pretreated with M1/Mdivi1 normothermically prior to 6‐h cold storage and warm reperfusion, followed by co‐culture with allogeneic CD8⁺ T cells, demonstrated by supernatant interferon gamma and granzyme‐B levels after 7 days of coculturing (n = 3). Surface expression of VCAM‐1 (C), MHC‐I (D), intracellular expression of Ki‐67 (E), and surface expression of PD‐L1 (F) at 6 h (top) and 24 h (bottom) post‐reperfusion in MCECs pretreated with M1/Mdivi1 normothermically prior to 6‐h cold storage and warm reperfusion (n = 3–6). (G) Abrogation of tolerogenic effect induced by pre‐cold storage M1/Mdivi1 treatment when PD‐L1 is blocked, demonstrated by supernatant interferon gamma and granzyme‐B levels after 7 days of coculturing in the presence of anti‐PD‐L1 blocking antibodies (n = 3). (****p < .0001, ***p < .005, **p < .01, *p < .05. Differences between untreated control and M1/Mdivi1‐treated groups, as well as between PD‐L1‐unblocked control and PD‐L1‐blocked groups, were analyzed using Student's t‐test. In panels B and G, interferon gamma and granzyme‐B levels were normalized to the number of ECs at the initiation of co‐culture to account for target differences)

FIGURE 4

Pharmacological modulation of mitochondrial fusion/fission in ECs alters the memory phenotype of co‐cultured T cells. Blue gating: Flow cytometry analyses showing the percentages of both effector memory population, with phenotype CD44⁺ CD62L⁻ (A) or CD44⁺ CCR7⁻ (B), and central memory population, with phenotype CD44⁺ CD62L⁺ (B), are lower in CD8⁺ T cells co‐cultured with pre‐cold storage M1/Mdivi1‐treated MCECs versus untreated controls (n = 3). Green gating: Flow cytometry analyses with a focus on the CD44^hi populations showing that both effector memory population, with phenotype CD44^hi CD62L⁻ (A) or CD44^hi CCR7⁻ (B), and central memory population, with phenotype CD44^hi CD62L⁺ (A) or CD44^hi CCR7⁺ (B), are lower in CD8⁺ T‐cells co‐cultured with pre‐cold storage M1/Mdivi1‐treated MCECs versus untreated controls (n = 3) (****p < .0001, ***p < .005, **p < .01, *p < .05. Differences between untreated control and M1/Mdivi1‐treated groups were analyzed using Student's t‐test)

FIGURE 5

Pharmacological modulation of mitochondrial fusion/fission in ECs mitigates the alloimmune response of co‐cultured T cells. (A) Flow cytometry analyses showing the percentages of proliferative CD8⁺ T cells, marked by Ki‐67 expression, are lower in T cells co‐cultured with pre‐cold storage M1/Mdivi1‐treated MCECs versus untreated controls (n = 3). (B) Flow cytometry analyses showing that both percentages of CD8⁺ T cells expressing the two cytotoxic proteins granzyme‐B and interferon gamma, and mean fluorescence intensity (MFI) of the cytotoxic proteins are lower in T cells co‐cultured with pre‐cold storage M1/Mdivi1‐treated MCECs versus untreated controls (n = 3). (**p < .01, *p < .05. Differences between untreated controls and M1/Mdivi1‐treated groups were analyzed using Student's t‐test)

FIGURE 6

Donor pre‐treatment with M1/Mdivi1 exerts mitochondria‐elongating effects on cardiac ECs but not cardiomyocytes. (A) Representative transmission electron microscopy images showing mitochondria in ECs but not in cardiomyocytes are morphologically elongated at the end of donor pre‐treatment with M1/Mdivi1. (B) Representative 3D confocal images showing mitochondria in ECs isolated from M1/Mdivi1‐pretreated donor hearts are elongated, and quantification of mitochondrial morphology performed as in Figure 1A (n = 3 mice/group with 50 cells/group, ****p < .0001, **p < .01. Differences between BD control and BD M1/Mdivi1 groups were analyzed using Student's t‐test)

FIGURE 7

Donor pre‐treatment with mitochondrial fusion promoter/fission inhibitor protects cardiac allografts from injury and mitigates T cell alloimmune response. (A) Cardiac injury, reflected by serum cardiac troponin I levels at 48 h posttransplant, is significantly reduced in mice receiving M1/Mdivi1‐pretreated allografts. (B) Histological staining and (C) quantification analysis showing significantly less cardiac injury and immune infiltration in M1/Mdivi1‐pretreated allografts versus untreated controls. (D) Brain‐dead donor delivery of M1/Mdivi1 significantly reduces innate immune cell infiltrates as demonstrated by myeloperoxidase immunohistochemistry in M1/Mdivi1‐pretreated allografts versus untreated controls. (E) Flow cytometry analyses showing no changes in effector memory (CD44⁺ CD62L⁻) and central memory (CD44⁺ CD62L⁺) compartments of CD8⁺ T cells in M1/Mdivi1‐pretreated allografts versus untreated controls. (F) CD8⁺ T cells infiltrating cardiac allografts have significantly lower granzyme‐B and interferon gamma production in M1/Mdivi1‐pretreated allografts compared to those infiltrating untreated controls, reflected by both frequencies of T cells producing these cytotoxic proteins and mean fluorescence intensity (MFI) of the proteins in T cells. (G) Flow cytometry analyses showing no changes in proliferative index (Ki‐67) in infiltrating CD8⁺ T cells (***p < .005, *p < .05. Differences between untreated controls and M1/Mdivi1‐treated groups were analyzed using Mann‐Whitney test for histological analysis and Student's t‐test for the remaining analyses)

FIGURE 8

Donor pre‐treatment with mitochondrial fusion promoter/fission inhibitor significantly prolongs cardiac allograft survival. (A) Schematics of the brain‐dead murine transplant model. (B) Percent survival of transplanted cardiac allografts showing donor pre‐treatment with M1/Mdivi1 after brain death leads to significantly improved survival outcomes (n = 4, p = .0091 by Mantel‐Cox test), an effect that is not observed with pre‐treatment of the antioxidant, N‐acetylcysteine, alone