In this study, we showed that high concentrations of ASCs in fat grafts promote fibrosis and decrease long-term retention. We also observed high levels of Th2 cells in the early stage and long-term persistence of M2 macrophages after fat grafting with excessive ASCs (Fig. 6). In addition, we found that ASCs can promote Th1–Th2 shifting in vitro (Fig. 7).
ASCs, which can be obtained from adipose tissue, have been experimentally shown to have angiogenic and adipogenic characteristics . In light of these functions, and because graft retention is mainly due to tissue regeneration, many studies have focused on the long-term retention of ASCs-assisted lipotransfer [14, 19, 20]. However, consisitent with the results of Paik and Natsuko’s research [16, 17], we also found that the concentrations of ASCs in adipose tissue influences the rate of retention. Also, we elevate concentrations of ASCs to 108 cells/ml in the study. Our result showed that addition of a suitable concentration of ASCs (104 cells/ml) into adipose tissue significantly improved long-term retention, whereas excessive ASCs (≥ 106 cells/ml) not only did not improve long-term retention but also significantly decreased long-term retention by increasing inflammation and fibrosis in the 108 group than other groups. Moreover, the number of macrophages in inflammatory cells of adipose tissue was significantly increased in the 108 group. This suggested that excessive ASCs in adipose tissue might lead to an increased macrophage inflammatory response that exacerbated fibrosis, thereby reducing the retention of fat grafts.
Macrophages play significant roles in tissue inflammation [21–25]. M1 (classically activated) macrophages mediate inflammatory responses, which are associated with high levels of pro-inflammatory cytokines [26, 27]. In fact, large numbers of macrophages infiltrated into the tissue after fat grafting. At the early stage of infiltration, mainly M1 macrophages occured to clear necrotic tissues and cells . Then, macrophages gradually transform from M1 to M2 (alternatively activated), which is a necessary process after fat grafting . While M2 macrophages could secrete anti-inflammatory factor and pro-angiogenic factor such as TGF-β and vascular endothelial growth factor (VEGF) to downregulate the level of inflammation and promote vascular growth into grafts which could recruit hematogenous stem cells to participate in the process of adipogenesis [30, 31]. Study showed that when M2 macrophages were added into grafts appropriately, fat grafting could be promoted . However, excess M2 macrophages can increase fibrosis inversely [29, 33]. M2 macrophages promote fibroblast proliferation and the expression of α-SMA, and α-SMA myofibroblast accumulation has been recognized as an early marker of tissue fibrosis . Indeed, M2 macrophages may be able to convert into fibroblasts . In this study, we observed a high expression of α-SMA and prolonged infiltration by M2 macrophages in the 108 group. However, the long-term presence of M2 macrophages may have promoted fibrosis , perhaps explaining the higher level of tissue fibrosis in the 108 group.
Interestingly, we found that the evolution of the Th2/Th1 ratio from weeks 1 to 12 was similar to that of the M2/M1 ratio from weeks 4 to 12 in all groups. This suggests that the Th1–Th2 shifting might promote the persistence of M2 macrophages after fat grafting. In fact, full macrophage activation requires two major signals in the context of the immune response, including the Th1 and Th2 responses . In naive T-helper cells, the IL-4 and IFN-γ genes are silent but can be activated to stimulate T-cells to begin to choose between the Th1 and Th2 cell fates [37, 38]. IFN-γ and IL-4 are produced by mutually inhibitory CD4 + T-helper cells: Th1 and Th2, respectively . In the Th1 response, innate IFN-γ induces the first wave of classical activation in M1 macrophages, stimulating IL-12 secretion, an important signal for Th1 activation. Upon Th1 activation, greater levels of IFN-γ induce long-lasting M1 macrophages; meanwhile, a full cytotoxic T-cell response is mounted. By contrast, in the Th2 response, IL-4 produced by Th2 cells induce a wave of alternative activation in M2 macrophages, which also provide signals that promote Th2 development [6, 39]. In addition, IL-10 secretion by M2 macrophages may also induce the development of repressor T-cells, which oppose Th1 activation . Meanwhile, a study published in SCIENCE pointed out a pro-regenerative response characterized by an mTOR/Rictor-dependent T helper 2 pathway that guides IL-4 dependent macrophage polarization is critical for tissue regeneration . Hence, the key process of M1 to M2 transformation of macrophages in fat grafting might be initiated by Th1–Th2 shifting. Th2 responses are essential for the control of extracellular parasites, including helminths, protozoa, and fungi, but they also contribute to allergy, increased susceptibility to other pathogens, and complications of infection such as fibrosis . For instance, Th2 cells can promote the M2 macrophages by upregulating arginase activity and increase L-ornithine, L-proline and polyamine concentrations, which promotes fibroblast proliferation, collagen production and ultimately fibrosis . Thus, the Th1–Th2 shifting might be necessary for adipose tissue regeneration, but the long-lasting high level of Th2/Th1 ratio might result in the long-term infiltration of M2 macrophage and fibrosis observed in the 108 group.
ASCs could regulate effector T-cell responses and have beneficial effects on various immune disorders [42, 43]. Moreover, ASCs can down-regulate IFN-γ and up-regulate IL-4, which could stimulate T-cells to begin to choose between Th1 and Th2 cell fates . Li et al. showed that the amount of IFN-γ production by Th1 cells is reduced by treatment with ASCs . In addition, Bassi et al. suggest that ASCs therapy could diminish the Th1 immune response . ASCs also potentially promote a Th2 shift in another research . In addition, Fiorina et al. administered allogeneic ASCs to NOD mice and observed a shift in Th1/Th2 cell balance towards Th2 cells . That is to say, the immunoregulatory capacity of ASCs might be related to these cells’ ability to promote the Th1–Th2 shift. We used the ASCs-CM and ASCs-Transwell models to explore the immunoregulatory capacity of ASCs and their ability to promote Th1–Th2 shifting in vitro, and the results also showed that when the concentrations of ASCs was up-regulated, the ratio of Th2%/Th1% increased. Moreover, at the same concentration, Th2%/Th1% was increased greater in the ASCs-transwell group than the ASCs-CM group after 24 hours suggesting that not only can the ASCs-CM promote the shift from Th1 to Th2, but also the continuous paracrine interaction between ASCs and CD4 + T-cells in vivo can promote Th1–Th2 shifting more promptly. Above all, due to the immunoregulatory capacity of ASCs, high concentrations of ASCs in adipose tissue can promote Th1–Th2 shifting, and the resulting excess of Th2 cells might promote the persistence of M2 macrophages and increase the level of fibrosis. Likely due to these phenomena, the long-term retention of fat grafting decreased in the 108 group.
By contrast, the ASCs in the 104 group played the opposite role. In these mice, long-term retention was higher than in the control group, and both fibrosis and the persistence of M2 macrophages were reduced. However, α-SMA expression did not differ between the 104 group and control group. Hence, we postulated that the immunoregulatory capacity of ASCs differed between the 104 group and the 108 group. Given that the α-SMA level was the same as in the control group, the main function of ASCs in the 104 group might be to inhibit excessive secretion of extracellular matrix (ECM) proteins and promote degradation of ECM proteins . In fact, there are several possible mechanisms of ASCs antifibrotic effects, including the regulation of TGF-β/Smad axis, the paracrine mechanisms, the antioxidant effects of ASCs and so on . Consequently, fibrosis was reduced, and retention was higher in the 104 group.
However, the surival capability of ASCs after transplantion was still uncertain. Some studies indicate that ASCs might just survive for a period time after grafting . By contrast, a tracing study revealed that intravenously injected ASCs, which were assumed to proliferate, were present in the graft until at least postoperative week 8 and mainly induced angiogenesis and adipogenesis by paracrine action rather than direct differentiation . Anyway, although dead ASCs might affect their immunoregulatory function, it would appear after the death of ASCs. While the paracrine effect of ASCs in the early stage after grafting is extremely definite. Whether or not ASCs died in the late stage of transplantation, its paracrine effect has a regulatory effect on CD4 + T-cell immune response, and both short-term and long-term paracrine effects of ASCs can promote Th1 to Th2 shifting. Similar results have been found in our experiments in vitro.
Based on our findings, it seems reasonable to conclude that in a clinical context, it is important to pay attention to the concentrations of ASCs in fat grafts. A suitable concentration of ASCs could decrease fibrosis and increase long-term retention, whereas excessive ASCs could have the opposite effects [51, 52]. Since ASCs can directly induce the phenotype of M2 macrophage, the transformation of macrophages may also affect the shifting process of CD4 + T-cells [6, 53]. The immunoregulation effect of ASCs in fat grafting may be due to promote transformation of both CD4 + T-cells and macrophages at the same time [44, 47, 54]. Thus, the transformation of CD4 + T-cells and macrophages might be a process of mutual promotion. We will further clarify the above in the next experiment. In addition, the immunoregulatory capacities of ASCs, i.e., inhibition of excessive ECM secretion, promotion of ECM degradation, and regulation of Th1–Th2 shifting, should be applied to the treatment of various diseases in the future.