Platelets Modulate Leukocyte Population Composition Within Perivascular Adipose Tissue

Abstract

Perivascular adipose tissue (PVAT) regulates vascular tone and is composed of adipocytes and several leukocyte subpopulations. Diet can modify PVAT function, as obesogenic diets cause morphological changes to adipocytes and skew the leukocyte phenotype, leading to PVAT dysregulation and impaired vasoregulation. Of note, platelets, the clot-forming cells, also modulate many facets of leukocyte activity, such as tissue infiltration and polarity. We aimed to determine whether platelets regulate the leukocyte populations residing within PVAT. Male C57Bl/6J mice were fed a Western diet (30% kcal sucrose, 40% kcal fat, 8.0% sodium) to develop obesogenic conditions for PVAT leukocyte remodeling. Diet was either administered acutely (2 weeks) or extended (8 weeks) to gauge the length of challenge necessary for remodeling. Additionally, platelet depletion allowed for the assessment of platelet relevance in PVAT leukocyte remodeling. Abdominal PVAT (aPVAT) and thoracic PVAT (tPVAT) were then isolated and leukocyte composition evaluated by flow cytometry. Compared to control, Western diet alone did not significantly impact PVAT leukocyte composition for either diet length. Platelet depletion, independent of diet, significantly disrupted PVAT leukocyte content with monocytes/macrophages most impacted. Furthermore, tPVAT appeared more sensitive to platelet depletion than aPVAT, providing novel evidence of platelet regulation of leukocyte composition within PVAT depots.

Keywords: Western diet; leukocytes; macrophages; monocytes; obesity; perivascular adipose tissue; platelets.

Figure 1

Analysis of broad leukocyte subsets in (A) aPVAT and (B) tPVAT following acute feeding and platelet depletion. Neither acute dietary modification nor treatment resulted in discernable changes when observing broad leukocyte classes in either PVAT depot. Leukocyte populations were identified by flow cytometry with pre-gating on the total, live, singlets and then CD45⁺ hematopoietic cells. Amongst CD45⁺ cells, myeloid cells were identified by CD11b positivity, T cells by CD3 positivity and B cells by B220 positivity. The percentage of cells are shown as a percentage of CD45⁺ cells.

Figure 2

Investigation of specific leukocyte subsets after acute feeding (2 weeks) and platelet-depleting antibody treatments. The proportions of CD4⁺ and CD8⁺ T cells relative to the total CD3⁺ T cell population remained unchanged in (A) aPVAT and (B) tPVAT, regardless of Western diet or platelet depletion. Within the subsets of CD11b⁺ myeloid cells, platelet depletion facilitated a trend toward increased Ly6G⁺ neutrophils (PMN) and reduced CD64⁺MERTK⁺ macrophages in both (C) aPVAT and (D) tPVAT. Platelet depletion trended toward a reduction in Ly6C⁺ monocytes in tPVAT only, while the combination of a Western diet and platelet depletion increased the percentage of Siglec-F⁺ eosinophils in both depots. For aPVAT and tPVAT myeloid cells, the percentage of myeloid cell subsets (i.e., neutrophils, eosinophils, monocytes and macrophages) are shown as a percentage of CD11b⁺ cells. The percentage of CD4+ and CD8+ T cells are shown as a percentage of CD3⁺ cells. * p (value) < 0.05.

Figure 3

Alterations in monocytes and macrophages that resulted from acute diet and platelet-depleting treatments. (A) Within aPVAT, an intact platelet population paired with a control diet reduced Ly6C^hiCX3CR1^lo classical and increased Ly6C^loCX3CR1^hi nonclassical monocytes, while platelet depletion paired with a Western diet reduced nonclassical monocytes. (B) Platelet depletion, regardless of diet, increased the percentage of classical monocytes and trended toward a reduction in the nonclassical monocytes in tPVAT. (C) Neither diet nor platelet depletion impacted the proportion of M1 or M2 macrophages within aPVAT. (D) Platelet depletion increased the proportion of M1 macrophages independent of diet. Regarding M2 macrophages in tPVAT, diet and treatment interventions had no effect. Monocytes and macrophages were identified by flow cytometry with pre-gating on the total, live, singlets and then CD45⁺CD11b⁺ cells. Amongst CD11b⁺Ly6G⁻Singlec-F⁻ cells, classical monocytes were defined as Ly6C^hiCX3CR1^lo and nonclassical monocytes were defined as Ly6C^loCX3CR1^hi. Amongst CD11b⁺ cells, the macrophages were defined as CD64⁺MERTK⁺ cells. * p (value) < 0.05.

Figure 4

Leukocyte subset disruption in aPVAT and tPVAT following 8-week feeding and platelet depletion treatments. (A) Within aPVAT, the combination of a Western diet and platelet depletion significantly increased the percentage of CD3⁺ T cells. A control diet and intact platelets trended toward increasing the percentage of CD11b⁺ myeloid cells, while platelet depletion alone trended toward increasing the percentage of B220⁺ B cells. (B) Platelet depletion significantly decreased the CD11b⁺ myeloid cell population within tPVAT, independent of diet. Additionally, a Western diet paired with platelet depletion increased the percentage of CD3⁺ T cells, while a Western diet alone trended toward reducing B220⁺ B cells. * p (value) < 0.05.

Figure 5

Lymphoid and myeloid cell subsets with 8-week feeding and platelet depletion treatment. (A) Within aPVAT, platelet depletion independent of diet resulted in an increase in CD4+ T cells, though the trend was more robust when paired with a Western diet. Inversely, the CD8+ population was diminished with platelet depletion coupled with a Western diet, with platelet depletion + control diet showing a similar trend. (B) Platelet depletion trended toward increasing the CD4+ population in tPVAT, withe Western diet pairing appearing more robust. Additionally, platelet depletion seemingly trended toward increasing the CD8+ population as well but did not yield a high degree of significance. (C) Platelet depletion coupled with a control diet increased the percentage of Siglec-F⁺ eosinophils in aPVAT. (D) Platelet depletion trends toward reducing the percentage of Ly6G⁺ neutrophils (PMN) in tPVAT. Additionally, an intact platelet population, with control diet feeding appeared to reduce tPVAT Siglec-F⁺ eosinophils. * p (value) < 0.05.

Figure 6

Frequency of monocyte and macrophage subsets in PVAT after 8-week extended feeding in conjunction with platelet depletion. (A) Control diet feeding coupled with intact platelets increased the percentage of Ly6C^hiCX3CR1^lo classical and conversely decreased the percentage of Ly6C^loCX3CR1^hi nonclassical monocyte subsets in aPVAT. Platelet depletion weakly trended toward a reduction in the percentage of classical monocytes. (B) Platelet depletion alone decreased the percentage of classical monocytes in tPVAT. Western diet alone reduced the percentage of nonclassical monocytes, with platelet depletion eliciting a trend toward increased nonclassical monocytes. Platelet depletion, regardless of diet, significantly reduced the percentage of CD11b⁺CD64⁺MERTK⁺iNOS⁺ M1 macrophages in both (C) aPVAT and (D) tPVAT. Inversely, platelet depletion trended toward increasing the percentage of CD11b⁺CD64⁺MERTK⁺CD206⁺ M2 macrophages in both adipose depots. * p (value) < 0.05.