Discussion
In this study, we used EAN mice to explore the effects of M2 macrophages and BAY11-7082 as an NF-κB inhibitor on this disease. Our results displayed that M2 macrophages ameliorated the clinical signs and reduced the duration of symptoms of EAN in mice by inhibiting pro-inflammatory M1 macrophages and cytokines accumulation. Additionally, the M2 macrophage polarization was increased in M2 treatment group, which was related to inhibition of NF-κB signaling pathway. BAY11-7082 could also diminish EAN symptoms by reducing the expression of TNF-α, IL-6 and IL-12, delaying the onset of EAN in preventive group. Further, BAY11-7082 greatly increased the percentage of M2 macrophage and reduced the percentage of M1 macrophage in preventive group. In vitro, BAY11-7082 inhibited the activity and proliferation of M1 macrophages. Overall, these results demonstrated the NF-κB signaling pathway may be involved in the pathogenesis of EAN by regulating the polarization of macrophages and inhibiting the expression of inflammatory cytokines. The results of adoptive transfer of M2 macrophages into EAN mice displayed the marked beneficial effects on EAN by inhibiting the activation of NF-κB.
Macrophages are broadly divided into two phenotypes: pro-inflammatory macrophages (M1) and anti-inflammatory macrophages (M2) [14]. M1 macrophages secrete pro-inflammatory cytokines that cause tissue damage and disease development, whereas M2 macrophages express high levels of anti-inflammatory molecule to reduce inflammation and promote disease recovery [14, 16]. Macrophages play either a pro-inflammatory or anti-inflammatory role in the different stages of GBS [17]. M1 cells promote the expression of major histocompatibility complex-II (MHC-II), adhesion molecules, reactive oxygen intermediates (ROI), and inflammatory cytokines, resulting inflammation, broken brood–nerve barrier (BNB), and demyelination [3]. In contrast, M2 macrophages exert a neuroprotective role in the pathogenicity of EAN [18]. M2 macrophages may contribute to the spontaneous re-myelination and regeneration of the axon [19, 20] by promoting T cell apoptosis, suppressing inflammatory responses[9], clearing myelin and axonal debris [16], and inducing the secretion of anti-inflammatory cytokines such as IL-10 and TGF-β[21].
In agreement with those findings, the present study showed that the improved outcome of EAN was associated with the higher proportion of M2 macrophages and the M2 macrophage polarization was increased in M2 treatment group. In addition, the role of BAY11-7082 inhibiting EAN clearly is related to shift macrophages from M1 to M2 type as the evidence with higher percentage of M2 macrophages and lower percentage of M1 macrophages in EAN treated by BAY11-7082.
NF-κB is an inducible transcription factor expressed in a large number of cells and involved in immune and inflammatory responses. It plays a critical role of cell differentiation, apoptosis as well as oncogenesis. NF-κB as a pro-inflammatory signaling pathway, facilitate the inflammatory reaction by up-regulation of NF-κB target genes encoding pro-inflammatory cytokines, chemokines and adhesion molecules. These signals lead to the recruitment and activation of neutrophils,macrophages and leukocytes to sites of inflammation [22].
NF-κB can modulate the inflammatory response in EAN as the several studies demonstrated that the the activated p65 of NF-κB was observed in peripheral nerve macrophages in the patients with AIDP or CIDP and EAN [23, 24]. The activated p65 of NF-κB in T cells and macrophages has higher intensity at the peak of EAN than control. The activation of NF-κB is induced by a large number of potent stimuli such as LPS, TNF-α and IL-1. Activated NF-κB is responsible for the expression of many pro-inflammatory cytokines (TNF, IL-1, IL-6 and IL-8), chemokines [25], adhesion molecules, prostaglandins, reactive oxygen species [26] and matrix metalloproteinases[27]. P65 is required for the leukocyte recruitment and the macrophages activation during the onset of inflammation [28]. However, recent studies have found that NF-κB activation promotes neuronal survival by inducing the transcription of anti-apoptotic genes and a number of growth factors [29-31]. P65/RelA over-expression induces the expression of anti-apoptotic gene and protects neurons from death. NF-κB protects neurons against amyloid β-peptide toxicity, glutamate-induced toxicity, and excitotoxic or oxidative stress [32].
In this work, we found that BAY11-7082 can improve EAN outcome by suppressing M1 macrophages and the expression of TNF-α, IL-6 and IL-12. Although our results indicated that inhibiting NF-κB enhanced polarization of M2 macrophages and repressed M1 macrophages, the underlying mechanisms are still unknown. In addition, previous studies have reported that inhibition of NF-κB can prolong the inflammatory process and maintain leukocyte activation [33]. Therefore, the possible effects that NF-κB mediate macrophages polarization in EAN, warrant further studies.