Macrophage Phenotype Induced by Circulating Small Extracellular Vesicles from Women with Endometriosis

Abstract

Evidence suggests that immune system dysfunction and macrophages are involved in the disease establishment and progression of endometriosis. Among the factors involved in this alteration in macrophage activity, Small Extracellular Vesicles (sEVs) have been described to play a role favoring the switch to a specific phenotype with controversial results. This study aims to investigate the potential effect of circulating sEVs in the plasma of well-characterized patients with endometriosis on the polarization of macrophages. sEVs were isolated from the plasma of patients diagnosed with endometriosis confirmed by histopathological analysis. Two groups of patients were recruited: the endometriosis group consisted of patients diagnosed with endometriosis by imaging testing (gynecological ultrasonography and/or magnetic resonance imaging), confirmed by histopathologic study (n = 12), and the control group included patients who underwent laparoscopy for tubal sterilization without presurgical suspicion of endometriosis and without endometriosis or signs of any inflammatory pelvic condition during surgery (n = 12). Human THP1 monocytic cells were differentiated into macrophages, and the effect of sEVs on cell uptake and macrophage polarization was evaluated by fluorescent labeling and measurement of the IL1B, TNF, ARG1, and MRC1 expression, respectively. Although no changes in cell uptake were detected, sEVs from endometriosis induced a polarization of macrophages toward an M2 phenotype, characterized by lower IL1B and TNF expression and a tendency to increase MRC1 and ARG1 levels. When macrophages were stimulated with lipopolysaccharides, less activation was also detected after treatment with endometriosis sEVs. Finally, endometriosis sEVs also induced the expression of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARG); however, treatment with rosiglitazone, a PPARG agonist, had no effect on the change in macrophage phenotype. We conclude that circulating sEVs in women with endometriosis have a certain capacity to shift the activation state of macrophages toward an M2 phenotype, but this does not modify the uptake level or the response to PPARG ligands.

Keywords: endometriosis; macrophages; small extracellular vesicles.

Figure 1

(A) Nanovesicle tracking assay confirms that the size of extracellular vesicles obtained corresponds to sEVs. (B) Western blot analysis of sEVs proteins CD9 and Alix from the different groups. Calnexin (CNX) was included as a negative control (the original is included in Supplementary Figure S1). (C) EV levels, expressed as μg prot/mL, detected in the plasma. C = Control; E = Endometriosis.

Figure 2

(A) Cell uptake of sEV by THP1 macrophages 2 h and 4 h (10×). (B) No relevant changes were observed in sEVs uptake by THP1 macrophages at 2 h or 4 h. (C) Confocal analysis of sEVs uptake (60×). sEVs were stained with red PKH26 dye and cells were stained with green PKH67 dye. C = Control E = Endometriosis. * = p < 0.05 vs. 2 h.

Figure 3

(A) Induction of IL1B, TNF, MRC1, ARG1, PPARG, and CD36 expression in THP1 macrophages treated with 10 µg/mL sEVs for 24 h. (B) Effect of sEVs on the expression of IL1B and MRC1 in THP1 macrophages treated with LPS (100 ng/mL). C = Control; E = Endometriosis. * = p < 0.05 vs. C.

Figure 4

Effect of pharmacological activation of PPARG on CD36 and macrophage phenotype using rosiglitazone treatment. C = Control; E = Endometriosis. * = p < 0.05 vs. C.