# Effects of _Cordycepps militaris_ Extracts on Macrophage as Immune Conductors
## 4 Discussion
Various compounds in phytoextracts such as melatonin and L-3-n-butylphthallide from celery and polyphenols from cocoa have been shown to activate the M2 polarization of macrophages [31; 32; 33]. Flow-cytometry and qPCR analysis in this research showed that the extracts significantly inhibited TNF-\(\alpha\) and IL-1\(\beta\) by suppression of M1 polarization in the macrophages (Figures 3 and 4). These results also suggest that the extracts strongly induced suppression of pro-inflammatory cytokines and activation of phagocytosis and contained compounds that activated M2 polarization. Macrophages also showed enhanced phagocytic activity dose-dependently. As per the t-SNE results, macrophages exposed to 1 mg/mL of the extracts formed populationswith strong activity (Figure 5c). This result suggests that the extracts caused the activation of M2 polarization and phagocytic activity of M2 macrophages. Activated macrophages modulated the differentiation of dendritic and T cells. First, activated macrophages strongly increased the number of CD11b\({}^{+}\) and Xcr1\({}^{+}\) DCs (Figure 5). From some reports [34; 35; 36], pre-conventional dendritic cells (pre-cDC) among DC precursors were differentiated by interferon regulatory factor 4 (IRF4), macrophage colony-stimulating factor receptor (M-CSFR), and granulocyte-macrophage colony-stimulating factor receptor (GM-CSF). In addition, IRF4 induces downregulation of pro-inflammatory cytokines in leukocytes [36]. Xcr1\({}^{+}\) DCs enhance T-cell survival and activation [37], while CD11b\({}^{+}\) DCs are involved in antigen presentation to CD4\({}^{+}\) cells associated with the induction of Th2 or Th17 cells [35; 38]. Furthermore, unlike LPS altered profile of inflammatory cytokines as increasing IL-18 and decreasing of IL-4 and IL-13 in macrophages, the extract enhanced the increase of IL-4 and 13 (Figure S1). These results suggest that the extract activates macrophages to synthesize suppressors of pro-inflammatory signals and enhances DC differentiation. In addition to stimulating CD304\({}^{+}\)FoxP3\({}^{+}\) T\({}_{\text{reg}}\) cells, the number of CD304\({}^{+}\)NK1.1\({}^{+}\) NKTL cells following incubation with conditioned medium from macrophages treated with the extract and LPS was 6.73 times higher than that observed when conditioned medium from LPS-challenged macrophages was used. FoxP3\({}^{+}\) T\({}_{\text{reg}}\) cells are known as homeostatic modulators of the immune system [39]. T\({}_{\text{reg}}\) cells play important roles in various immune systems, including hypersensitivity suppression, pathogenic immunopathology, and autoimmune disease suppression [40]. These results show that the extract activates macrophages to enhance the differentiation of CD304\({}^{+}\)NK1.1\({}^{+}\) NKTL cells and suppress the inflammatory response induced by LPS. Although the _C. millitaris_ extract 1 \(\upmu\)g/mL contained lower quantities of cordycepin than did the conditional medium from cordycepin-treated macrophages, cell differentiation was enhanced 1.6 times by the extract conditioned medium compared to the cordycepin conditioned medium. Additionally, in the results for comparison of CD3/CD28 and the extract stimulation (Figure S7), the stimulating capacity was evaluated similarly between them. These results suggest that the extract contained other bioactive compounds in addition to cordycepin that stimulated the differentiation of immune cells. Moreover, the extract is more effective to activate an immune response when compared to cordycepin. With a mouse's cell line, this research documented that macrophages are an immune conductor under the extract. However, in vivo study for the extract will be considered to prove the activation of macrophage as an immune conductor by the extract. If results from in vivo research correspond to the results of this research, the extract is applied to a bioactive material in functional foods, cosmetic, and immunological additive for stock feeds.