# Effects of _Cordycepps militaris_ Extracts on Macrophage as Immune Conductors
## 4 Discussion  
Although there are many research studies that documented immunological functions of cordycepin and _C. militaria_s extract [7], they are poorly understood for immunological modulation by macrophage under the extract. Cordyceps has been used as a healthy food because of its various effects of cleansing the blood, suppressing harmful bacteria and inflammation, and improving immunity [23]. Cordycepin, a functional substance of _Cordyceps sinensis_, was first found in _C. militaria_ fruiting bodies [24]. In addition, 3\({}^{\prime}\)-deoxyadenosine has many biological activities, such as anti-inflammatory, antioxidative and anti-aging, antitumor, anti-cancer, and anti-leukemia activities [8]. LPS, one of the components of the cell wall of gram-negative bacteria, induces a strong inflammatory response through toll-like receptor 4 (TLR4) activation in host cells and sepsis. LPS is important for inflammatory reactions in host cells, and it plays a role in protecting bacterial cells from external toxic substances and antibiotics. The total free amino acid, adenosine, and cordycepin contents were 83.35 mg/g, 0.24%, and 1.33%, respectively, in the _C. militaria_ hydrolytic extract [25]. The concentration of cordycepin was higher than that of adenosine [25]. The concentrations of adenosine and 3\({}^{\prime}\)-deoxyadenosine in the extracts were found to be 0.05 \(\pm\) 0.01 mg/mL and 0.47 \(\pm\) 0.09 mg/mL, respectively, using HPLC. The cytotoxic concentrations of cordycepin in various cell lines ranged from 300 \(\upmu\)M to 400 \(\upmu\)M [26]. Although the extracts in this study contained low contents of cordycepin, the cytotoxic concentration (CC\({}_{50}\)) of the extract was established at these low concentrations. This result means that other compounds excluding cordycepin affect attenuation of cellular viability in macrophages. Recently, the relationship between inflammation and chronic disease has been reported. Although inflammation acts as a protective barrier against bacteria in the early stages of damage, it causes various chronic diseases such as obesity, diabetes, cancer, and brain and heart diseases [27]. Macrophages derived from monocyte precursors undergo specific differentiation depending on the local tissue environment. They respond to environmental cues within tissues such as cell damage, activated lymphocytes, or microbial products to differentiate into distinct functional phenotypes [28]. Macrophages are polarized into two phenotypes in response to various factors--classical macrophages M1 and alternative macrophages and M2 [15; 26]. M1 macrophages are typically activated by IFN-\(\gamma\) or LPS and produce pro-inflammatory cytokines, IL-1\(\beta\), and TNF-\(\alpha\), to kill pathogens. However, excessive production of M1 macrophages is associated with the onset of chronic inflammatory diseases [15]. M2 macrophages are selectively activated by exposure to specific cytokines such as IL-4, IL-10, or IL-13, and are involved in wound healing and tissue repair with anti-inflammatory activity [18; 19]. M1 and M2 cells secrete major cytokines, and the process of the production of these cytokines has the histopathological characteristics of various inflammation-related diseases. Although the M1 phenotype is essential for the immune system, prolonged M1 polarization leads to several diseases [29]. The M2 phenotype, on the other hand, attenuates inflammation through phagocytosis of a pathogen [15; 30]. In this study, the extracts dose-dependently suppressed the M1 polarization of macrophages but activated M2 polarization. Interestingly, qPCR showed that the levels of TNF-\(\alpha\) and IL-1\(\beta\) were notably downregulated in the macrophages at 1 mg/mL of the extracts (Figure 2). Furthermore, unlike LPS altered cytokine profile such as increasing IL-18 and decreasing of IL-4 and IL-13 in macrophages, the extract enhanced increasing of IL-4 and 13 (Figure S1).