New factors in heart failure pathophysiology: Immunity cells release of extracellular vesicles

Abstract

Leukocyte-shed extracellular vesicles (EVs) can play effector roles in the pathophysiological mechanisms of different diseases. These EVs released by membrane budding of leukocytes have been found in high amounts locally in inflamed tissues and in the circulation, indicating immunity cell activation. These EVs secreted by immune cell subsets have been minimally explored and deserve further investigation in many areas of disease. In this study we have investigated whether in heart failure there is innate and adaptive immune cell release of EVs. Patients with chronic heart failure (cHF) (n = 119) and in sex- and age-matched controls without this chronic condition (n = 60). Specifically, EVs were quantified and phenotypically characterized by flow cytometry and cell-specific monoclonal antibodies. We observed that even in well medically controlled cHF patients (with guideline-directed medical therapy) there are higher number of blood annexin-V⁺ (phosphatidylserine⁺)-EVs carrying activated immunity cell-epitopes in the circulation than in controls (p < 0.04 for all cell types). Particularly, EVs shed by monocytes and neutrophils (innate immunity) and by T-lymphocytes and natural-killer cells (adaptive immunity) are significantly higher in cHF patients. Additionally, EVs-shed by activated leukocytes/neutrophils (CD11b⁺, p = 0.006; CD29⁺/CD15⁺, p = 0.048), and T-lymphocytes (CD3⁺/CD45⁺, p < 0.02) were positively correlated with cHF disease severity (NYHA classification). Interestingly, cHF patients with ischemic etiology had the highest levels of EVs shed by lymphocytes and neutrophils (p < 0.045, all). In summary, in cHF patients there is a significant immune cell activation shown by high-release of EVs that is accentuated by clinical severity of cHF. These activated innate and adaptive immunity cell messengers may contribute by intercellular communication to the progression of the disease and to the common affectation of distant organs in heart failure (paracrine regulation) that contribute to the clinical deterioration of cHF patients.

Keywords: chronic heart failure; extracellular microvesicles (EVs); extracellular vesicle (EV); immune cells; inflammation; liquid biopsies.

FIGURE 1

Cellular origin and distribution of AV⁺ EVs in cHF and controls. (A) Distribution of EVs from total leukocytes (CD45⁺), T-lymphocytes (CD3⁺ and CD3⁺/CD45⁺), neutrophils (CD15⁺), monocyte/macrophage (CD16⁺) and natural-killer cells (CD56⁺). (B) Distribution of EVs from activated neutrophils (CD29⁺/CD15⁺) and from activated leukocytes (CD11b⁺). A p < 0.05 was considered significant (U Mann–Whitney test). AV⁺, annexin V⁺; cHF, chronic heart failure; EVs, extracellular microvesicles; LEVs, leukocyte-derived EVs; lEVs, lymphocyte-derived EVs; mEVs, monocyte-derived EVs; nEVs, neutrophil-derived EVs; NKC-EVs, natural-killer cells-derived EVs; PFP, platelet-free plasma.

FIGURE 2

Distribution of AV⁺-EVs of immune-cell origin considering disease severity according to NYHA. (A) Distribution of EVs from total leukocytes (CD45⁺), T-lymphocytes (CD3⁺ and CD3⁺/CD45⁺), activated leukocytes (CD11b⁺) and from activated neutrophils (CD29⁺/CD15⁺). A p < 0.05 was considered significant (U Mann–Whitney test). (B) ROC curve analyses to evaluate EVs and NT-proBNP association to NYHA severity, with AUC indicated along its 95% CI. A p < 0.05 was considered significant. AUC, area under the curve; AV⁺, annexin V⁺; cHF, chronic heart failure; CI, confidence interval; EVs, extracellular microvesicles; LEVs, leukocyte-derived EVs; lEVs, lymphocyte-derived EVs; nEVs, neutrophil-derived EVs; NYHA, New York Heart Association; PFP, platelet-free plasma; ROC, receiver operating characteristic curve.

FIGURE 3

Distribution of AV⁺-EVs from immune-cell origin considering disease etiology. (A) Distribution of EVs from T-lymphocytes (CD3⁺), activated leukocytes (CD29⁺) and from activated neutrophils (CD29⁺/CD15⁺). A p < 0.05 was considered significant (U Mann–Whitney test). (B) ROC curve analyses were used to evaluate the impact of EVs and combinations of EVs with NT-proBNP on HF severity (NYHA) in ischemic patients (AUC indicated along its 95% CI). A p < 0.05 was considered significant. AUC, area under the curve; AV⁺, annexin V⁺; cHF, chronic heart failure; CI, confidence interval; EVs, extracellular microvesicles; LEVs, leukocyte-derived EVs; lEVs, lymphocyte-derived EVs; nEVs, neutrophil-derived EVs; NYHA, New York Heart Association; PFP, platelet-free plasma; ROC, receiver operating characteristic curve.

FIGURE 4

Distribution of AV⁺-EVs of immune-cell origin considering disease severity in ischemic patients. Distribution of EVs of total leukocytes (CD45⁺), T-lymphocytes (CD3⁺ and CD3⁺/CD45⁺), activated leukocytes (CD11b⁺) and from activated monocytes (CD11b⁺/CD14⁺) and activated neutrophils (CD29⁺/CD15⁺). A p < 0.05 was considered significant (U Mann–Whitney test). AV⁺, annexin V⁺; EVs, extracellular microvesicles; LEVs, leukocyte-derived EVs; lEVs, lymphocyte-derived EVs; mEVs, monocyte-derived EVs; nEVs, neutrophil-derived EVs; NYHA, New York Heart Association; PFP, platelet-free plasma.