Depletion of γδ T Cells Leads to Reduced Angiogenesis and Increased Infiltration of Inflammatory M1-like Macrophages in Ischemic Muscle Tissue

Abstract

γδ T cells, a small subset of T cells in blood, play a substantial role in influencing immunoregulatory and inflammatory processes. The functional impact of γδ T cells on angiogenesis in ischemic muscle tissue has never been reported and is the topic of the present work. Femoral artery ligation (FAL) was used to induce angiogenesis in the lower leg of γδ T cell depleted mice and wildtype and isotype antibody-treated control groups. Gastrocnemius muscle tissue was harvested 3 and 7 days after FAL and assessed using (immuno-)histological analyses. Hematoxylin and Eosin staining showed an increased area of tissue damage in γδ T cell depleted mice 7 days after FAL. Impaired angiogenesis was demonstrated by lower capillary to muscle fiber ratio and decreased number of proliferating endothelial cells (CD31⁺/BrdU⁺). γδ T cell depleted mice showed an increased number of total leukocytes (CD45⁺), neutrophils (MPO⁺) and neutrophil extracellular traps (NETs) (MPO⁺/CitH3⁺), without changes in the neutrophils to NETs ratio. Moreover, the depletion resulted in a higher macrophage count (DAPI/CD68⁺) caused by an increase in inflammatory M1-like macrophages (CD68⁺/MRC1^-). Altogether, we show that depletion of γδ T cells leads to increased accumulation of leukocytes and M1-like macrophages, along with impaired angiogenesis.

Keywords: NETs; angiogenesis; gamma delta T cells; ischemia; macrophage polarization; macrophages; neutrophil extracellular traps; neutrophils; proliferation; γδ T cells.

Figure 1

γδ T cell depleted mice show increased ischemic tissue damage. (a) Representative H&E pictures of gastrocnemius muscles of wildtype (WT) (top), isotype antibody-treated (ISO) (middle) and γδ T cell depleted mice (bottom) 7 days after femoral artery ligation (aFAL). Scale bars: 1000 µm. (b) Detailed images of the black boxes shown in (a). Scale bars: 100 µm. (c) The scatter plot shows the area of ischemic tissue damage (%) of WT, ISO and TCRγδ T cell depleted mice 7 days aFAL. Data are means ± SEM, n = 5 per group. * p < 0.05, ns ≥ 0.05 (WT vs. ISO vs. TCRγδ depletion) by one-way ANOVA with the Tukey’s multiple comparisons test.

Figure 2

Absence of γδ T cells decreases angiogenesis. Scatter plots show the number of (a) endothelial cells (CD31⁺/CD45⁻) and (b) proliferating endothelial cells (CD31⁺/CD45⁻/BrdU⁺) per muscle fiber of ischemic gastrocnemius muscles of WT, ISO and γδ T cell depleted mice 7 days after femoral artery ligation (aFAL). Data are means ± SEM, n = 5 per group. * p < 0.05, ns ≥ 0.05 (WT vs. ISO vs. TCRγδ T cell depletion) by one-way ANOVA with the Tukey’s multiple comparisons test. (c) Representative images of ischemic gastrocnemius muscles of WT (top), isotype antibody-treated (middle) and TCRγδ T cell depleted mice (bottom) 7 days aFAL. Single channel pictures (small images) show endothelial cells (CD31, gray) and proliferating cells (BrdU, red). Merged images also show leukocytes (CD45, green) and nuclei (DAPI, blue). Scale bars: 30 µm.

Figure 3

γδ T cell depletion leads to reduced activation of VEGF receptor 2 (Tyr1175). (a) The scatter plot shows the number of VEGF receptor 2 (Tyr1175)/CD31 double-positive endothelial cells (Phospho-VEGF receptor 2 (Tyr1175)⁺/CD31⁺) per muscle fiber in ischemic gastrocnemius muscles of WT, ISO and TCRγδ T cell depleted mice 3 days after femoral artery ligation (aFAL). Data are means ± SEM, n = 5 per group. * p < 0.05, ns ≥ 0.05 (WT vs. ISO vs. TCRγδ depletion) by one-way ANOVA with the Tukey’s multiple comparisons test. (b) Representative images of ischemic gastrocnemius muscles of wildtype (WT, top), isotype antibody-treated (ISO, middle) and TCRγδ T cell depleted mice (TCRγδ depl., bottom) 3 days aFAL. The VEGF receptor 2 was stained with an antibody recognizing the activated phospho-VEGF receptor 2 (Tyr1175) form (green). Endothelial cells were stained with an antibody against CD31 (white), while nuclei were labeled using DAPI (blue). Scale bars: 30 µm.

Figure 4

Mice lacking γδ T cells showed increased accumulation of leukocytes in ischemic muscle tissue. (a) Representative immunofluorescence images of analyzed gastrocnemius muscles of WT (top), isotype antibody-treated (middle) and TCR γδ T cell depleted mice (bottom) 7 days after femoral artery ligation (aFAL). Leukocytes were stained with an antibody against CD45 (green), while nuclei were labeled using DAPI (blue). Scale bars: 50 µm. (b) The scatter plot shows the absolute number of leukocytes (CD45⁺) per mm² in ischemic gastrocnemius muscle tissue from WT, ISO and TCR γδ T cell depleted mice 7 days aFAL. Data shown are means ± SEM, n = 5 per group, a defined area of 1.5 mm² of ischemic muscle was analyzed per mouse. * p < 0.05, ns ≥ 0.05 (WT vs. ISO vs. TCR γδ depletion) by one-way ANOVA with the Tukey’s multiple comparisons test.

Figure 5

Absence of γδ T cells results in increased accumulation of neutrophils without affecting the formation of neutrophil extracellular traps. The scatter plots show (a) the total number of neutrophils (myeloperoxidase; MPO⁺ cells) per mm² (upper plot), (b) the occurrence of NETs (MPO⁺/CitH3⁺ (citrullinated histone 3)) per mm² (middle plot), and (c) the relative proportion of NETs positive MPO⁺ cells of all MPO⁺ cells (lower plot) in the ischemic gastrocnemius muscle tissue of mice of the WT, isotype antibody-treated and γδ T cell depleted group 3 days after femoral artery ligation (aFAL). Data are means ± SEM, n = 5 per group. * p < 0.05, ns ≥ 0.05 (WT vs. ISO vs. TCR γδ depletion) by one-way ANOVA with the Tukey’s multiple comparisons test. A defined area of 1.5 mm² of ischemic gastrocnemius muscle tissue was analyzed per mouse. (d) Representative immunofluorescence images of ischemic gastrocnemius muscles from WT (top), ISO (middle) and TCR γδ T cell depleted mice (bottom) 3 days aFAL. Images show neutrophils (MPO⁺/DAPI⁺), NETs (MPO⁺/CitH3⁺/DAPI⁺) and nuclei (DAPI⁺) labeled with anti-MPO (red), anti-CitH3 (green) and DAPI (blue). Scale bars: 20 µm.

Figure 6

Depletion of γδ T cells leads to an increased number of M1-like polarized macrophages. Scatter plots show the absolute number of (a) all macrophages (CD68⁺) per mm² (top plot), (b) MRC1-negative macrophages (CD68⁺/MRC1⁻ (mannose receptor C-type 1)) (middle plot) and (c) MRC1-positive macrophages (CD68⁺/MRC1⁺) (bottom plot) in ischemic gastrocnemius muscle tissue of wildtype (WT), isotype (ISO) and TCR γδ T cell depleted mice 7 days aFAL. Data are means ± SEM, n = 5 per group. * p < 0.05, ns ≥ 0.05 (WT vs. ISO vs. TCR γδ depletion) by one-way ANOVA with the Tukey’s multiple comparisons test. (d) Representative immunofluorescence images of analyzed ischemic muscle tissue of WT, ISO and TCR γδ T cell depleted mice. Macrophages (CD68⁺/DAPI⁺) were stained with antibodies targeting CD68 (green), MRC1 (red) and DAPI (blue). CD68⁺/MRC1⁻/DAPI⁺ cells were defined as M1-like polarized macrophages and CD68⁺/MRC1⁺/DAPI⁺ cells as M2-like polarized macrophages. Scale bars: 30 µm.