Discussion
Macrophages are prominent innate immune cells and divided into M1 and M2 subtypes, and new research also found that M3 switch phenotype also exists[22,23]. Previous studies showed that increased macrophages were associated with neuronal damage in MS: In the acute phase, M1 macrophage were activated, along with the release of large amounts of pro-inflammatory cytokines, neuro-inflammation, CNS demyelination, and neuronal death[24-26]. However, as the disease progressed, M2 macrophages increased gradually, and the maximum levels of M2 and M1 macrophage reduced gradually from the peak of disease to the remission of EAE. In the late stages of the disease, M2 macrophages played a dominant role in the CNS and released a variety of anti-inflammatory cytokines, which reduced the inflammatory response and promoted tissue repair, while inhibiting the further progression of EAE[25]. Our results is consistent with the results of previous studies[27].
Recent studies have shown that some treatment for MS like glucocorticoids, fingolimod, glatirameracetate, teriflunomide and interferon-beta can downregulate expression M1 macrophage or activate M2 macrophage[28-32]. Previous studies have found that therapy with M2 macrophage or microglia can prevent the occurrence of type 1 diabetes and the motor neurons damage caused by spinal cord injured, and also alleviate EAE symptoms in rats[33,13,14]. However, there are the limited macrophages source and needed to expand more methods to obtain enough macrophages. In recent years, researchers have applied cytokines and compounds that can stimulate the differentiation of bone marrow stem cells into M2 macrophages, which have proven to be effective in transforming stem cells into M2 macrophage. In our study, IL-4 and M-CSF could successfully induce bone marrow stem cells to differentiate into M2 macrophages that have been transferred into EAE mice at the early stage of the disease. In comparison with the control group, the M2-treated group showed simultaneous development of symptoms, but their peak symptoms were alleviated, and symptom relief was faster. The degrees of spinal cord inflammation and the degrees of inflammation and demyelination in spinal codes and inflammatory factors secretion in blood were lower in M2-treated group than those in control group, which is similar to other results[34,12]. However, the difference from the previous studies is that our experiment is using the M2 macrophages induced from bone marrow stem cells and inject them by intravascular route, which will greatly facilitate future clinical operations and make cell therapy more likely. Our experiment also found that after treatment with M2 macrophage in EAE mice, the macrophages in the spleen were decreased, which included both M1 and M2 macrophages, however, the M2 macrophage decreased more significantly. Pro-inflammatory factors are decreased and anti-inflammatory factors are increased in peripheral blood.
It has been evidenced that once the inflammatory reaction leads to the immune system activation, and then the immune system tries to maintain the internal environment in balance by various ways. In our study, after a large number of M2 macrophages injected into EAE mice, the declined macrophage activation in the spleen of EAE mice through reducing M2 macrophages mainly was for restoring internal environment homeostasis. At the same time, due to the infusion of a large number of M2 macrophages, anti-inflammatory factors were significantly increased in the blood, promoting repairing the damaged tissue and alleviating the sings of EAE.
NF-κb is a key intracellular signal transduction molecule that can regulate the expression of many molecules involved in the early stage of immune and inflammatory responses. Induced NF-κb is mainly in the form of p50/p65 isodimers which p65 played a major role[15,16], and that is involved in the occurrence and development of MS. Previous research findings implied that changes in the activity or protein expression of NF-κb are closely related to the severity of EAE and the incidence of disease, clinical fraction, and inflammation of the CNS were all shown to decrease in mice with NF-κb defects[17,18]. Moreover, NF-κb also plays a central role in activating and differentiating T cells and blocking NF-κb function is an effective method to prevent autoimmune encephalitis[35].
After we applied M2 macrophages, the symptoms of the mice were reduced, and NF-kb expression in the spinal cord was also reduced too. Therefore, we wondered whether NF-kb was involved in the polarization of macrophages to M1 and M2 cells. Studies have shown that the NF-κB signal transduction pathway participates in the inflammatory immune response induced by macrophage[36]. NF-κB activity is closely related to the number, state, degree, and product of microglial activation, that is, the higher the NF-κB activity, the more activated microglial cells and the more inflammatory factors[37]. Therefore, we hypothesized that blocking NF-kb pathway can reduce the polarization of M1 macrophages, increase the number of M2 macrophages, and reduce the symptoms of EAE.
We applied a NF-κb blocker BAY-11-7082, which can block the phosphorylation of NF-κb65, and observed the effects on M1 and M2 macrophages in vitro and in vivo . The results showed that the severity of EAE was alleviated after the application of the blocker, and the therapeutic effect in the prevention group was better than that in the treatment group, indicating that the effect of early blockade was more obvious. The analysis of macrophages in the spleen of mice at the peak of EAE displayed that the proportion of total macrophages was lower in the NF-κb blockade group(prevented and treated group) than the control group, in which the proportion of M1 macrophage declined significantly, however, the proportion of M2 decreased a bit. Meanwhile,in vitro experiments revealed that the blocker blocked the activity of both M1 and M2 macrophages simultaneously. However, the blockade of activity of M1 was more obvious than activity of M2, which is similar to in vivo study. Our results suggest that inhibition of NF-κb pathway can inhibit the generation of macrophages, especially M1 macrophage and pro-inflammatory factors. Therefore, early addition of this inhibitor can block the pro-inflammatory effect of M1 more effectively and reduce the inflammatory response in EAE mice.
The present study demonstrates that (a) macrophages are involved in the occurrence and development of EAE disease: M1 macrophages have pro-inflammatory effects, and M2 macrophages have anti-inflammatory effects; (b)bone marrow stem cells could be successfully induced to differentiate into M2 macrophages; (c) M2 macrophages blocked the NF-κb pathway and had a promising therapeutic effect on EAE, which enable rational therapeutic approaches for MS and other autoimmune diseases in the future; (d)NF-κb blockade effectively prevented EAE development and alleviated the severity of EAE via blockading NF-κb pathway. In the future, we will further study how the NF-κb pathway involving in the process of macrophage polarization.