To investigate the effect of MSCs exosomes (mesenchymal stem cells derived exosome, MSCs-Exo) activated byToll-like receptors 3 and 4 signaling pathways on the polarization of macrophages.

After cultured MSCs sourced fromrat bone marrow by differential adherence, exosomesfrom MSCs, MSCs activated by TLR3 signaling and MSCs activated by TLR4 signaling were extracted from cell culture supernatant by exosome Extraction Kit. M0 macrophages cultured with RPMI-1640 containing 10%FBS and 10%L929 medium,were divided into 6 groups: a control group, the MSCs-Exo, MSCs-Exo activated by TLR4 signaling pathways, MSCs-Exo activated by TLR3 signaling pathways, LPSand IL-4+IL-13. After cultured for 48 h, morphology of macrophages was observed under an electronic microscope. The immunophenotype (CD206, Arg-1, TNF-α and iNOS ) and difference of expression of inflammatory factors (CCL22, IL-1β, IL-6 and IL-10) of macrophages in each group were detected by flow and qPCR, respectively.Single factor analysis of variance and independent t test were used for statistical analysis.

The cultured MSCs are identical with the characteristics of mesenchymal stem cells. The results showed that MSCs-Exo was a double layer membrane vesicle structure with the diameter of 40 ~ 200 nm, expressing ofexosomes protein CD9 and HSP70. The morphology of macrophages in each group was observed under an electronic microscope, Morphology of long carboxylic shape and many pseudopods were observed in groups withmacrophages stimulated by MSCs-Exo, MSCs-Exo activated by TLR4 signaling pathways and MSCs-Exo activated by TLR3 signaling pathways. High expression of CD206 (107.2±6.87, 102.4±9.83, 112.0±9.24 vs 56.0±7.38, F = 47.234, P = 0.000, respectively) and Arg-1 (135.2±6.87, 130.2±7.59, 203.4±9.07 vs 117.8±9.12, F = 109.827, P = 0.009, 0.048, 0.000), and low expression of TNF-α (27.0±5.65, 24.6±5.02, 25.6±4.15 vs 36.6±7.09, F = 4.882, P = 0.046, 0.015, 0.017) were respectively detected by flow cytometry in the groups withthe macrophages activated by MSCs-Exo, MSCs-Exo activated by TLR4 signaling pathways and MSCs-Exo activated by TLR3 signaling pathways, and low expression ofiNOS (240.2±8.43 vs 308.8±9.88, P = 0.000)with the macrophages activated by MSCs-Exo,and invariant expression of iNOS (P > 0.05) with the macrophages activated by MSCs-Exo activated by TLR4 signaling pathways and MSCs-Exo activated by TLR3 signaling pathways.High expression of CCL22 (2.277±0.744, 1.570±0.209, 1.642±0.443 vs 1.000±0.111, F = 23.654, P = 0.015, 0.003, 0.031) and IL-10 (1.233±0.136, 2.426±0.343, 1.390±0.155 vs 1.000±0.130, F = 103.251, P = 0.048, 0.000, 0.008), low expression ofIL-1β (0.383±0.035, 0.640±0.143, 0.242±0.073 vs 1.000±0.082, F = 12.315, P = 0.000, 0.005, 0.000)and IL-6 (0.386±0.066, 0.655±0.046, 0.533±0.090 vs 1.000±0.204, F = 30.140, P = 0.001, 0.006, 0.016) were detected by qPCR.

Polarization of macrophages to M2 type can be stimulated by MSCs-Exo activated by TLR3 and TLR4 signaling pathway.