Immune modulation of macrophage response by Cordyceps militaris extracts

Backgrounds: Cordyceps militaris is a well-known medicinal fungus. Cordycepin, a metabolite of this fungus, has strong biological activities against leukemia, oxidative stress, aging, tumors, and inflammation. Methods: HPLC analysis was conducted to measure the content of corydycepin in the extract. Real time PCR was performed to evaluate the cytokines. Immunoactivity including the polarization, phagocytic activity and cellular differentiations were evaluated by flow cytometry. Results: The yields of cordycepin and adenosine in the extract were 11.75 µg and 1.25 µg (per gram fresh mycelium), respectively. From measurements of the bioactivity in the extract, the levels of TNF-α and IL-1β in macrophages treated with lipopolysaccharides (LPS) were found to be approximately 4 and 48 times higher than those in the control, as shown by qRT-PCR. However, cells treated with 1 µg/mL of the extract showed 13 and 10-fold lower TNF-α and IL-1β levels when compared to LPS-treated cells. This was corroborated by flow-cytometry, where their levels were 20 and 14 times lower, respectively. Addition of the extract to LPS-treated cells enhanced M2 polarization and attenuated M1 polarization. In addition, the extract also dose-dependently activated macrophage phagocytosis. Under treatment with the extract conditioned medium, DCs were strongly differentiated toward CD11b + and Xcr1 + cells as their density were 13.6 and 6.26 times higher than those in the LPS conditioned medium, respectively. Moreover, the number of T reg and NKT cells differentiated in the extract conditioned medium were increased about 1.67 and 6.73 times than those in the LPS conditioned medium, respectively. Conclusions: These results suggest that the C. militaris hydrolytic extract strong effects on

is a precursor of polysaccharides, ergosterol, mannitol, and vitamins [3]. Cordycepin is not produced by all Cordyceps species; it was first discovered in 1951 by Professor Cunningham of the University of Glasgow, United Kingdom, in C. militaris [4]. The pharmacological functions of cordycepin are very diverse; it shows anti-cancer, anti-inflammatory, anti-viral, anti-leukemia, anti-tumor, anti-diabetic, and anti-obesity effects, and is known to modulate the human immune system [5][6][7][8]. Macrophages, derived from monocytes, play an important role in phagocytosis in disease lesions and act as antiinflammatory antigen-presenting cells [9]. They also drive homeostasis as part of innate and acquired immunities. During inflammatory reactions, they also play a defensive role by producing nitric oxide (NO) and cytokines [10]. Lipopolysaccharides (LPS), major endotoxins, are derived from gramnegative bacterial cell walls [11]. Macrophages exposed to LPS produce inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF), and polarize to M1 macrophages [12,13]. Although previous studies have shown the anti-inflammatory effects of C. militaris extracts [5][6][7][8], there is no clear evidence whether these extracts could act on macrophages to modulate the immune response. The goal of this study was to quantify the 3'-deoxyadenosine levels in C. militaris and to evaluate the biological functions of C. militaris extracts in immunological modulation of LPStreated macrophages.

qPCR analysis
Total RNA was extracted from cells using Ribospin mini kit (GeneAll, Seoul, Korea); then RNA samples were converted to cDNA using DiaStarTM 2X RT Pre-mix kit (Solgent, Daejeon, Korea) at 55 °C for 1 h and then at 95 °C for 5 min. Real time-PCR was performed using Topreal ™ qPCR 2X PreMIX (Enzynomics; Daejeon, Korea) with the cDNA samples and analyzed using the StepOnePlus Real-Time PCR System instrument (Thermo Fisher Scientific Inc, Germering, German). TNF-α, IL-1β, and b-actin primers ( Table 1) were purchased from Bioneer (Seoul, Republic of Korea). PCR conditions were as follows: initial denaturation of 15 min at 95°C, followed by 35 cycles of 10 s at 95°C, 15 s at 60°C, and elongation of 30 s at 72°C. Data were analyzed using the StepOne Software V2.3.

Phagocytic activity
After treatment with LPS and C. militaris extracts, macrophages were exposed to fluorescencelabelled E. coli particles and phagocytosis was examined using Vybrant™ Phagocytosis Assay Kit (Thermo Fisher scientific Inc, Germering, Germany) for 2 h. Stained macrophages were analyzed by flow cytometry (BD calibur and FlowJo 10.6.1, BD science).

Statistical analysis
The data were analyzed by performing paired t-test and variance (ANOVA) using SPSS software V26.0 (IBM, New York, USA). P values (P < 0.05) were calculated using Turkey's method.

Measurement of cordycepin levels in Cordyceps militaris extracts.
Cordycepin and adenosine levels were quantified by HPLC using appropriate standards. The total cordycepin and adenosine concentrations in the extracts were 0.47 ± 0.09 and 0.05 ± 0.01 µg/mL, respectively. Reported to the mycelium weight, the concentrations of cordycepin and adenosine in the fresh mycellium were 11.75 µg/g and 1.25 µg/g, respectively.

Effects of Cordyceps militaris extracts on the expression of IL-1β and TNF-α in macrophages
The mRNA levels of pro-inflammatory cytokines TNF-α and IL-1β were quantified in RAW 264.7 cells treated with LPS and extracts containing cordycepin. In macrophages stimulated only with LPS, the mRNA levels of TNF-α and IL-1β increased approximately 4-to 5-folds compared with the control (Fig. 2a). For macrophages treated with LPS and cordycepin extracts, the mRNA levels of TNF-α and IL-1β dramatically decreased, especially for 1 µg/mL of extract (Fig. 2a). Fluorescence in situ hybridization results also revealed that TNF-α and IL-1β were downregulated dose-dependently in macrophages treated with these extracts (Fig. 2b). Notably, treatment with 1 µg/mL cordycepin extract dramatically downregulated these cytokine levels (Fig. 2C3).

Polarization of macrophages
For cells not treated with the cordycepin extract, the number of M1 macrophages increased approximately four times following stimulation with LPS; however, under LPS treatment, the number of M2 macrophages decreased by approximately 60% compared to the control (Fig. 3). Surprisingly, when treated with C. militaris extracts, the number of M1 macrophages decreased and that of M2 macrophages increased dose-dependently (Fig. 3). When 1 µg/mL extract was used, the number of M1 macrophages decreased approximately 4 times and the number of M2 macrophages increased approximately 9 times compared to the number observed following LPS treatment (Fig. 3).

Phagocytic activity of macrophages
Changes in the phagocytic activity was not clear when macrophages were treated only with LPS; however, when cells were treated with LPS and cordycepin extracts, their phagocytic activity increased dose-dependently ( Fig. 4a, b). Result of t-SNE showed that most cells had a weak phagocytic activity, but when treated with 1 µg/mL cordycepin extract, macrophages showed a strong phagocytic activity (Fig. 4c). When macrophages were treated with 0.1 µg/mL cordycepin extract, their phagocytic activity was dramatically stimulated compared to that of cells treated only with LPS.
Moreover, when cells were treated with 1 µg/mL cordycepin extract, the phagocytic activity of macrophages increased 13 times compared to that observed following treatment with LPS only (Fig. 4d).

Differentiation patterns of monocytes and T lymphocytes
Although LPS blocked the differentiation of CD11b + and Xcr1 + cells from monocytes, following treatment with the conditioned medium from macrophages treated with cordycepin and LPS, dendritic cells (DCs) were strongly differentiated toward CD11b + and Xcr1 + cells as their densities increased 13.6 and 6.26 times, respectively, compared to those observed following treatment with conditioned medium from LPS-stimulated macrophages (Fig. 5). Compared to cordycepin, the extract conditioned medium slightly enhanced their differentiation from monocytes than differentiation by the cordycepin conditioned medium (Fig. 5). The conditioned medium from macrophages treated with LPS and cordycepin stimulated the differentiation of T cells to regulatory T cells (T reg ) and natural killer T (NKT) cells (Fig. 6). Notably, the differentiation of NKT cells increased by 6.75 times compared to that observed following treatment with conditioned medium from macrophages stimulated with LPS only (Fig. 6c, d). Moreover, although T cells were stressed with LPS, the differentiation of T reg and NKT cells following treatment with conditioned medium from cordycepin-treated macrophages was increased about 1.67 and 6.73 times, respectively compared to that observed when treated with conditioned medium from LPS-stimulated macrophages (Fig. 6b, d).

Discussion
Cordyceps has been used as a health food because of its various effects of cleansing the blood, suppressing harmful bacteria and inflammation, and improving immunity [15]. Cordycepin, a functional substance of Cordyceps sinensis, was first found in C. militaris fruiting bodies. 3'-Deoxyadenosine has many biological activities such as anti-inflammatory, antioxidative and antiaging, antitumor, anti-cancer, and anti-leukemia activities [5]. Total free amino acid, adenosine, and cordycepin contents were 83.35 mg/g, 0.24%, and 1.33%, respectively, in the C. militaris hydrolytic extract. The concentration of cordycepin was higher than that of adenosine [16]. The concentrations of adenosine and 3'-deoxyadenosine in the extracts were found to be 0.05 ± 0.01 µg/mL and 0.47 ± 0.09 µg/mL, respectively, using HPLC. The cytotoxic concentrations of cordycepin in various cell lines ranged from 300 to 400 µM [17]. Although the extracts in this study contained low contents of cordycepin, the cytotoxic concentration of the extract was established at these low concentrations. These results suggested that the extract contained various compounds, and all these compounds, except cordycepin, could affect cellular apoptosis.
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 [18].
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 [19].
Macrophages are polarized to two phenotypes in response to various factors: classical macrophages, M1 and alternative macrophages, M2 [9,17]. M1 macrophages are typically activated by IFN-γ or LPS and produce pro-inflammatory cytokines, IL-1β and TNF-α, to kill pathogens. However, excessive production of M1 macrophages is associated with the onset of chronic inflammatory diseases [9]. 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 [12,13]. 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 [20]. The M2 phenotype, on the other hand, attenuates inflammation through phagocytosis of a pathogen [9,21]. 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-α and IL-1β were notably downregulated in the macrophages at 1 µg/mL of the extracts (Fig. 2). Various compounds in phytoextracts such as melatonin and L-3-n-butylphthalide from celery and polyphenols from cocoa have been shown to activate the M2 polarization of macrophages [22][23][24]. Flow-cytometry and qPCR analysis showed that the extracts significantly inhibited TNF-α and IL-1β by suppression of M1 polarization in the macrophages. These results also suggested 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 µg/mL of the extracts formed populations with strong activity (Fig. 4c).
This result suggested 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 (Fig. 5). From some reports [25][26][27], 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 down-regulation of pro-inflammatory cytokines in leukocytes [27]. Xcr1 + DCs enhance T cell survival and activation [28], while CD11b + DCs are involved in antigen presentation to CD4 + cells associated with the induction of Th2 or Th17 cells [26,29]. These results suggest that the cordycepin extract activates macrophages to synthesize suppressors of pro-inflammatory signals and enhances DC differentiation.
Besides stimulating CD304 + FoxP3 + T reg cells, the number of CD304 + NK1.1 + NKT 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-stimulated macrophages was used. FoxP3 + T reg cells are known as homeostatic modulators of the immune system [30]. T reg cells play important roles in various immune systems, including hypersensitivity suppression, pathogenic immunopathology, and autoimmune disease suppression [31]. These results show that the extract activates macrophages to enhance the differentiation of CD304 + NK1.1 + NKT and suppress the inflammatory response induced by LPS. Moreover, although the C. militaris extract 1 µg/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. 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.

Conclusions
Consequently Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   Lymphocyte differentiation by activated macrophages in the extract conditioned medium