Changes in the spectrum of extracellular vesicles produced by THP-1 cells during polarization toward M1 or M2 macrophages

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Abstract

BACKGROUND: Macrophages are capable of secreting extracellular vesicles that exert a wide range of biological effects, including modulation of the immune response under pathological conditions.

AIM: The work aimed to compare the qualitative and quantitative composition of extracellular vesicles produced by THP-1 cells depending on the concentration and duration of activation with phorbol 12-myristate 13-acetate and the direction of polarization toward M1 or M2 macrophages.

METHODS: THP-1 cells were activated with different concentrations of phorbol 12-myristate 13-acetate (100 and 10 ng/mL). Polarization toward M1 macrophages was induced using IFN-γ and LPS, and toward M2 using IL-4 and IL-13. Cells and their extracellular vesicles were immunophenotyped for CD80, CD64, HLA-DR, CD206, CD209, and CD163. Relative gene expression levels of IL-1β, IL-6, IL-8, IL-12p40, TNFα, CXCL10, CD163, CD206, CCL22, IL-10, FN, and GAPDH were assessed. The size and concentration of extracellular vesicles were measured by nanoparticle tracking analysis. The protein composition of extracellular vesicles was additionally assessed for the presence of tetraspanin receptors (CD9, CD63, CD82, and CD81) and flotillin-1.

RESULTS: Activation of cells with high doses of phorbol 12-myristate 13-acetate followed by polarization toward M1, compared to M2, led to increased expression of CD80, CD209, and CD163. Regardless of the applied activation–polarization protocol, THP-1 cells were distributed into distinct, compact clusters according to the results of discriminant analysis of gene expression levels. Activation was accompanied by a more than 10-fold increase in extracellular vesicle production. High-dose phorbol 12-myristate 13-acetate activation followed by M1 polarization resulted in secretion of the highest number of extracellular vesicles (188×108 [185×108; 202.5×108] particles/mL), of larger size (134 ± 6.1 nm), and expressing CD63 and CD82. However, their flotillin-1 content was reduced.

CONCLUSION: Thus, high-dose phorbol 12-myristate 13-acetate activation of THP-1 cells is more effective for subsequent polarization. Depending on the applied polarization protocol, cells produce extracellular vesicles differing in both quantity and composition.

About the authors

Darina B. Sambur

Almazov National Medical Research Center

Email: sambour-darina@mail.ru
ORCID iD: 0009-0009-6352-813X
SPIN-code: 8885-9190
Russian Federation, Saint Petersburg

Olga V. Kalinina

Almazov National Medical Research Center

Email: olgakalinina@mail.ru
ORCID iD: 0000-0003-1916-5705
SPIN-code: 7752-7929

Dr. Sci. (Biology)

Russian Federation, Saint Petersburg

Arthur D. Aquino

Almazov National Medical Research Center

Email: akino97@bk.ru
ORCID iD: 0000-0001-6516-7184
SPIN-code: 3395-5556

Dr. Sci. (Biology)

Russian Federation, Saint Petersburg

Polina V. Tirikova

Almazov National Medical Research Center

Email: tipo.paulina2002@yandex.ru
ORCID iD: 0000-0002-4433-1640
Russian Federation, Saint Petersburg

Kseniya D. Zubkova

Almazov National Medical Research Center

Email: zubkowa.ksenija@gmail.com
ORCID iD: 0009-0007-8108-0311
Russian Federation, Saint Petersburg

Irina I. Dreizis

Almazov National Medical Research Center

Email: ira.dreyzis@gmail.com
ORCID iD: 0009-0004-3673-015X
Russian Federation, Saint Petersburg

Artem A. Rubinstein

Almazov National Medical Research Center; Institute of Experimental Medicine

Email: arrubin6@mail.ru
ORCID iD: 0000-0002-8493-5211
SPIN-code: 6025-1790
Russian Federation, Saint Petersburg; Saint Petersburg

Andrey S. Trulioff

Almazov National Medical Research Center; Institute of Experimental Medicine

Email: trulioff@gmail.com
ORCID iD: 0000-0002-7495-446X
SPIN-code: 8688-7506

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg; Saint Petersburg

Igor V. Kudriavtsev

Almazov National Medical Research Center; Institute of Experimental Medicine

Email: igorek1981@yandex.ru
ORCID iD: 0000-0001-7204-7850
SPIN-code: 4903-7636

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg; Saint Petersburg

Alexey S. Golovkin

Almazov National Medical Research Center

Author for correspondence.
Email: golovkin_a@mail.ru
ORCID iD: 0000-0002-7577-628X
SPIN-code: 8803-2425

MD, Dr. Sci. (Medicine)

Russian Federation, Saint Petersburg

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3. Fig. 1. Microscopy of THP-1 cells depending on the activation and polarization protocol: native THP-1 cells (a); exposure to 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 h (protocol 1) (b); exposure to 10 ng/mL PMA for 24 h followed by a 24 h rest period (protocol 2) (c). M0_1, cells activated according to protocol 1; M1_1, cells polarized toward the M1 macrophage phenotype after activation according to protocol 1; M2_1, cells polarized toward the M2 macrophage phenotype after activation according to protocol 1; M0_2, cells activated according to protocol 2; M1_2, cells polarized toward the M1 macrophage phenotype after activation according to protocol 2; M2_2, cells polarized toward the M2 macrophage phenotype after activation according to protocol 2.

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4. Fig. 2. Immunophenotyping of THP-1 cells activated according to protocol 1 (a) and protocol 2 (b), followed by polarization toward M1 and M2 macrophages. Group designations as in Fig. 1.

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5. Fig. 3. Relative expression of IL-1β, IL-6, IL-8, IL-12p40, TNFα, and CXCL10 genes in intact THP-1 cells, activated according to protocol 1 (a) and protocol 2 (b), followed by polarization toward M1 and M2 macrophages. Group designations as in Fig. 1. *р < 0.05, **р < 0.01, ***p < 0.0001.

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6. Fig. 4. Relative expression of CD163, CD206, CCL22, IL-10, and fibronectin (FN) genes in intact THP-1 cells, activated according to protocol 1 (a) and protocol 2 (b), followed by polarization toward M1 and M2 macrophages. Group designations as in Fig. 1. *р < 0.05, **р < 0.01, ***p < 0.0001.

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7. Fig. 5. Discriminant analysis based on the relative expression levels of 11 genes in intact THP-1 cells, activated according to protocol 1 (a) and protocol 2 (b), followed by polarization toward M1 and M2 macrophages. The plot shows clusters formed by cells of the respective groups (THP-1, M0_1, M1_1, M2_1, and M0_2, M1_2, M2_2). Each point represents one sample.

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8. Fig. 6. Presence of tetraspanin receptors (CD9, CD63, CD82, and CD81) and surface molecules CD163, CD206, HLA-DR, and CD209 characteristic of THP-1 cells according to protocol 1 (a) and protocol 2 (b), followed by polarization toward M1 and M2 macrophage phenotypes. Group designations as in Fig. 1.

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9. Fig. 7. Western blot performed with antibodies against flotillin-1. Group designations as in Fig. 1.

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