• 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • br Decreasing the levels of catecholamines reduces


    3.2. Decreasing the levels of catecholamines reduces M2-polarized macrophages
    To evaluate the interaction between catecholamines and the im-mune system, we further explored the activation states of splenic and intratumoral macrophages in the 6OHDA-treated mice. Although there was little impact on the polarization of splenic macrophages (Fig. 2A, upper, P = 0.19 for H446, P = 0.95 for HCC827), flow cytometry re-sults suggested that the reduction in catecholamine levels increased the percentage of M1-polarized macrophages significantly and maintained a higher ratio of M1/M2 in tumors (Fig. 2A, lower). The mean M1/M2 ratios reversed from 0.28 to 1.14 in H446 mice and from 0.24 to 1.02 in HCC827 mice after 6OHDA treatment.
    In order to verify the influence of increased M1/M2 ratio on the tumor microenvironment, we assessed the levels of several classical cytokines of activated macrophages in removed tumor tissues. IL-6, IL-10, and IL-12 are typical macrophage-associated factors and can reflect different activation states. While IL-6 and IL-12 are secreted by M1-polarized macrophages and regarded as the pro-inflammatory factors, IL-10 is discharged by M2-polarized macrophages as an im-munosuppressive molecule. As expected, we observed a remarkable decrease in IL-10 levels in tumor tissues, but the drop in IL-6 (P = 0.21 for H446, P = 0.06 for HCC827) and IL-12 (P = 0.42 for H446, P = 0.17 for HCC827) levels was not significant (Fig. 2B). These ob-servations seem to support the idea that catecholamines participate in re-educating the phenotypes and activation states of tumor-associated macrophages and decreased catecholamine levels could trigger a re-duction in the percentage of M2-polarized macrophages. 
    3.3. Adrenergic-stimulated macrophages tend to exhibit an M2-like phenotype
    The finding that decreased catecholamine levels correlated with increased M1/M2 radio led us to hypothesize that catecholamines might promote macrophages’ shift toward the M2-side. To explore the role of catecholamines in macrophage polarization, BMDMs were iso-lated from mice and stimulated toward M0 or M1, and then incubated with EPI or NE. Flow cytometry results revealed that catecholamines not only increased the percentage of M2-polarized macrophages in M0 KPT-330 slightly to 5.2% in EPI groups and 8.4% in NE groups (Fig. 3A, upper), but also strongly promoted a shift in M1 cells toward M2 be-cause the percentage rose to 16.4% and 23.5% respectively (Fig. 3A, lower).
    To highlight a stronger switch to M2 by M1 cells than by M0 cells, we examined the impact of catecholamines on M1 macrophages in the following experiments. Consistent with the above data, the transcrip-tion of M1-associated gene iNOS was inhibited (Fig. 3B, upper), con-comitant with the increased M2-associated gene Arg-1 in catechola-mine-treated M1 macrophages (Fig. 3B, lower). An analysis of cytokines also consolidated our hypothesis, M1-associated IL-6, IL-12, TNF-a re-duced significantly and M2-associated IL-10 markedly increased after catecholamine treatment (Fig. 3C). Taken together, these findings support the notion that adrenergic-stimulated macrophages display an M2-polarized phenotype.
    Y. Xia, et al. Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
    Y. Xia, et al. Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
    Fig. 4. Catecholamine stimulation promotes tube formation in vitro and tumor neovascularization in vivo. (A) HUVECs (green) were co-incubated with M1 mac-rophages, EPI- or NE-treated M1 macrophages, and M2 macrophages (red). Tube formation assays were conducted after 6 h to measure their capillary-like tubular structures. Representative photomicrographs are shown (scale bar: 200 μm); n = 3. The number of junctions, branches and tubules, total length, and total tubules area was measured in M1-, M2-polarized macrophages and catecholamine-treated M1 cells. (B) In vivo, the density of microvessels and M2 macrophages were obtained from the immunohistochemistry of serial tumor sections (scale bar: 100 μm); n = 3. *p < 0.05. **p < 0.01. ***p < 0.001. Abbreviations: N.S.: non-sta-tistical significance; A.U.: arbitrary units. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) r> 3.4. Adrenergic-stimulated macrophages contribute to angiogenesis
    Considering that M2-polarized macrophages are often connected with tumor angiogenesis, we investigated the role of adrenergic-sti-mulated macrophages on vascular density and vasculature patterns in vitro and in vivo. Firstly, the M1 and M2 cells were set as two controls. Next, M1 cells treated with catecholamines for 24 h were collected and co-incubated with HUVECs to evaluate the tube formation capacity in vitro. From M1, EPI-treated M1, and NE-treated M1 to M2 cells, sta-tistical analyses of capillary patterning revealed a gradually increased tendency in tube junctions, branches, length, and lumens (Fig. 4A), consistent with their position in the M1–M2 spectrum. In vivo, the distributions of M1 and M2 macrophage cells were compared and linked to microvascular density (MVD). We found that there were few iNOS-stained M1 cells in tumor areas but lots of CD163-stained M2 cells