GMI, a fungal immunomodulatory protein, ameliorates SARS-CoV-2 envelope protein-induced inflammation in macrophages via inhibition of MAPK pathway

Abstract

Clinically, COVID-19 is often accompanied by a severe immune response (cytokine storm) which produces a large number of cytokines, such as TNF-α, IL-6 and IL-12, and consequently causes acute respiratory distress syndrome (ARDS). GMI is a type of fungal immunomodulatory protein that is cloned from Ganoderma microsporum and acts as modulating immunocyte for various inflammatory diseases. This study identifies GMI as a potential anti-inflammatory agent and determines the effects of GMI on the inhibition of SARS-CoV-2-induced cytokine secretion. Functional studies showed that SARS-CoV-2 envelope (E) protein induces inflammatory process in murine macrophages RAW264.7 and MH-S cells and in phorbol 12-myristate 13-acetate (PMA)-stimulated human THP-1 cells. GMI exhibits a strong inhibitory effect for SARS-CoV-2-E-induced pro-inflammatory mediators, including NO, TNF-α, IL-6, and IL-12 in macrophages. GMI reduces SARS-CoV-2-E-induced intracellular inflammatory molecules, such as iNOS and COX-2, and inhibits SARS-CoV-2-E-stimulated phosphorylation of ERK1/2 and P38. GMI also downregulates pro-inflammatory cytokine levels in lung tissue and serum after the mice inhale SARS-CoV-2-E protein. In conclusion, this study shows that GMI acts as an agent to alleviate SARS-CoV-2-E-induced inflammation.

1. Introduction

The pandemic coronavirus disease 2019 (COVID-19) that has swept the world since December 2019 is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection depends on the interaction between spike protein of SARS-CoV-2 and the angiotensin-converting enzyme 2 (ACE2) in host cells [1]. If human cells or tissues are infected with SARS-CoV-2, a series of reactions are induced in the body, including virus production, initiation of immune response, and the release of many mediators, to fight the infection. Clinically, severe COVID-19 induces systemic hyper-inflammation, known as a cytokine storm, which causes acute respiratory distress syndrome (ARDS), multiple organ failure and can result in death [2], [3]. Increasing evidence shows that patients with COVID-19-induced ARDS have higher mortality rates than those who do not exhibit ARDS-related symptoms [4], [5]. Alleviating the cytokine storm in patients with COVID-19 is an effective clinical strategy.

A cytokine storm is a clinical condition that is characterized by uncontrolled hyper-inflammation that is caused by activated immune cells which overproduce various inflammatory cytokines [6]. Many studies show that high levels of pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, IL-8 and IL-12, and chemokines such as interferon (INF) and monocyte chemoattractant protein-1 (MCP-1) create a cytokine storm in patients with severe COVID-19, which results in damage to multiple organs and can result in death [2], [7], [8]. Therefore, blocking these inflammatory mediators may be an effective treatment for severe COVID-19. Currently, traditional anti-inflammatory drug glucocorticoid is commonly used to treat patients with SARS-CoV-2 infection because it inhibits NF-κB signals, which reduces the production of inflammatory factors [9]. Cytokine or cytokine receptor antagonists may offer additional clinical benefits. For example, IL-1 signaling blockade by canakinumab and anakinra improves respiratory function in COVID-19 patients with ARDS [10], [11]. Siltuximab, sarilumab, and tocilizumab that target IL-6 signaling may reduce the severity of infection and mortality in severe COVID-19 patients [12]. Etanercept blocks the TNF-α cassette to reduce the release of excessive cytokine release and hyperinflammation [13]. A blockade of cytokines-mediated downstream JAK/STAT signaling using baricitinib and ruxolitinib may also alleviate inflammation in patients with severe COVID-19 [14], [15]. However, there is still no suitable drug in clinical practice yet. Development of a safe and effective drug to control the SARS-CoV-2-induced cytokine storm and balance the immune responses is important. Notably, herb derivatives can be repurposed to reduce inflammatory molecules and reduce the cytokine storm that is caused by SARS-CoV-2 infection [16].

Since the first fungal protein was isolated from the mushroom Ganoderma lucidum mycelium in 1989 [17], many fungal proteins have been discovered. These identified fungal proteins have highly conserved amino acid sequences and structures and have been proven to regulate immune cells, so they are called fungal immunomodulatory proteins (FIPs) [18], [19]. Increasing evidence shows that FIPs exhibit anti-inflammation and anti-tumor functions so they have a high potential for development as a drug [19]. GMI, which is a FIP, is isolated and cloned from Ganoderma microsporum. Many studies determine the effects of GMI on anti-cancer activity but not on immunomodulatory functions because GMI suppresses tumor progression by inducing autophagy [20], [21]. Therefore, further exploring the immunomodulatory efficacy of GMI is an important research topic.

The authors previously showed that GMI prevents SARS-CoV-2 infection via a multifunctional broad-spectrum, such as the induction of ACE2 degradation of host cells, the inhibition of cell fusion, and the elimination of ACE2 binding with spike protein [22], [23]. This study determines the anti-inflammatory effects and the mechanism of GMI in SARS-CoV-2 infection. The inflammation that is induced by the SARS-CoV-2 subunits in macrophages and lung tissue of mouse is measured and experiments are performed to determine whether GMI inhibits SARS-CoV-2-related inflammatory molecules and to determine the mechanism for the inhibition of a cytokine storm by GMI.

2. Materials and methods

2.1. Materials

GMI (Cat: 767593), dissolved in sterilized PBS (1 mg/mL), was obtained from MycoMagic Biotechnology Co., Ltd. (New Taipei, Taiwan) [22]. LPS (E. coli O55:B5) was purchased from Sigma-Aldrich. SARS-CoV-2-envelope (E) protein (NBP2–90986) was purchased from Novus Biologicals (CO, USA). SARS-CoV-2-spike (S) protein (SPN-C52H9) was purchased from ACROBiosystems (DE, USA). Phorbol 12-myristate 13-acetate (PMA; P-1039-1MG) was purchased from AG Scientific (CA, USA). Dexamethasone was purchased from Cayman Chemical (Ann Arbor, MI, USA). PD98059 (ERK inhibitor; 167869-21-8) and SB203580 (P38 inhibitor; 152121-47-6) were purchased from AG Scientific (CA, USA).

2.2. Cell lines and cell culture

RAW264.7 (murine macrophages), MH-S (mouse alveolar macrophages) and THP-1 (human monocyte) cells were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). RAW264.7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, GIBCO-Life Technologies) that was supplemented with 10 % heat-inactivated fetal bovine serum (FBS, HyClone, MA, USA) and penicillin/streptomycin (100 units/mL; Biological Industries, Cromwell, CT). MH-S and THP-1 cells were Roswell Park Memorial Institute (RPMI) 1640 medium (GIBCO-Life Technologies) that was supplemented with 2 mM l-glutamine, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 10 % heat-inactivated FBS. All of the adherent cells were detached by incubation with trypsin-EDTA (Invitrogen, Co., CA, USA). The cells were maintained in a humidified 5 % CO₂ atmosphere at 37 °C.

2.3. Cell viability assay

Macrophages (2 × 10⁴ cells) were seeded into 96-well plates and incubated for 24 h to determine the cytotoxic effects of GMI. Macrophages were stimulated with LPS (100 ng/mL), SARS-CoV-2-E (1 μg/mL) or SARS-CoV-2-S (1 μg/mL) in the presence or absence of different concentrations of GMI (0–1.2 μM) for 24 h. After incubation, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye was added to each well and incubation lasted for 4 h. The cell viability was measured as previously described [24].

2.4. NO production assay

Macrophages (2 × 10⁴ cells/well in 96-well for 24 h incubation) were simultaneously stimulated with LPS (100 ng/mL), SARS-CoV-2-E (1 μg/mL) or SARS-CoV-2-S (1 μg/mL) in the presence or absence of GMI (0–1.2 μM) for 24 h. Nitric oxide (NO) production was measured by a Griess assay using the method of a previous study [25], [26]. Individual LPS-induced NO production was designated as 100 % for each experiment.

2.5. Enzyme-linked immunosorbent assay (ELISA) for cytokines

Macrophages (2 × 10⁴ cells in 96-well plates) were treated with various concentrations of GMI (0–1.2 μM) and vehicle (PBS) for 30 min, followed by LPS (100 ng/mL), SARS-CoV-2-E (1 μg/mL) or SARS-CoV-2-S (1 μg/mL) for 24 h. The levels of TNF-α, IL-6, IL-1β, IL-12 and IFN-γ in the cultured medium of macrophages and lung epithelial cells were measured using an ELISA kit (BioLegend, CA, USA) according to the manufacturer's instructions. A series of dilutions of various cytokines (ranging from 0 to 500 pg/mL) were used as standard curves for each experiment. Data was collected by detecting A450 nm and A570 nm (as reference absorbance) using a TECAN Sunrise™ ELISA Reader (Tecan Group Ltd., Männedorf, Switzerland). Individual LPS-induced production of cytokines was designated as 100 % for each experiment.

2.6. Western blot assay

Cells (5 × 10⁵ cells) were seeded in 6-cm cell culture dishes for 24 h. Cells were then treated with LPS (100 ng/mL) or SARS-CoV-2-E (1 μg/mL), followed by GMI (0, 0.3 and 0.6 μM) for 24 h. After treatment, cells were harvested and rinsed with cold PBS containing 1 % Na₃VO₄ and lysed using a specific lysis buffer with protease inhibitor [27]. Cell lysates were collected by centrifugation at 13,000 ×g for 10 min at 4 °C. Cell lysates (30 μg) were separated on 10 % SDS-PAGE and specific molecules were detected using a western blot analysis, using the method of a previous study [28]. Antibodies against COX-2 (SC-1747) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Antibodies against iNOS (GTX74171) and tubulin (GTX112141) were purchased from GeneTex (Hsinchu, Taiwan). Antibodies against phosphorylated JNK (J4750), ERK1/2 (M8159) and P38 (M8177) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

2.7. Animal model

Male C57BL/6 mice (6–8 weeks) were used for an in vivo study and were purchased from the National Laboratory Animal Center (NLAC, Taipei, Taiwan). The mice were isolated for at least 1 week before experimental manipulations and the procedure was approved by the NYCU Institutional Animal Care and Use Committee (IACUC Approval NO: 1101212). The mice were divided into 6 h and 24 h exposure groups and were exposed to PBS, GMI, or SARS-CoV-2-E for 30 min (Fig. 5A). GMI (100 μg) and SARS-CoV-2-E (20 μg) were dissolved in physiologic saline (1 mL). A nebulizer (Aerogen AG-AP1000, Aerogen Ltd. Galway, Ireland) was used to administer GMI (100 μg/mL) and SARS-CoV-2-E (20 μg/mL) to the mouse at a flow rate of 0.25 mL/min. The mice were placed in a 2.8 L cage and exposed to the GMI and SARS-CoV-2-E aerosol for 30 min. After exposure, the mice were sacrificed and the lung tissues and serum were harvested and stored at −80 °C. The deposited doses of GMI and SARS-CoV-2-E are calculated as [9]:

$$\text{Deposited Dose}\left(\frac{\mathrm{\mu g}}{\mathrm{kg}}\right)=\frac{\mathit{Concentration}\left(\frac{\mathit{\mu g}}{L}\right)\times \mathit{Minute\; Volume}\left(\frac{L}{\mathit{min}}\right)\times \mathit{IF}\times \mathit{DF}}{\mathit{Body\; Weight}\left(\mathit{kg}\right)}.$$

The concentration of GMI in air is 100 μg/2.8 L cage volume and the concentration of SARS-CoV-2-E in air is 20 μg/2.8 L cage volume. The respiratory minute volume is calculated as 0.021 L/min for mice. If there are no non-respirable particles in the aerosol, the inhalable fraction (IF) is 1, the deposition fraction (DF) is 0.055, which is related to the aperture of the nebulizer (3 μm), and the average body weight of the mice is 0.02 kg. The deposited doses of GMI for 30 min of exposure are 61.875 μg/kg, and the deposited doses for SARS-CoV-2-E for 30 min of exposure are 12.375 μg/kg. The procedure is that for a previous study [22].

2.8. Statistical analysis

All data is expressed as mean ± SD for more than three time-independent experiments. Statistical differences between each experimental group are determined using a t-test using GraphPad Prism8. The threshold for statistical significance is *P < 0.05, **P < 0.01 and ***P < 0.001.

3. Results

3.1. Envelope protein of SARS-CoV-2 stimulates inflammatory responses of macrophages

Studies show that SARS-CoV-2 subunits induce inflammatory cytokines such as IL-6 and TNF-α [29], [30]. This study initially determined whether SARS-CoV-2 subunits (SARS-CoV-2-Envelop (E) and SARS-CoV-2-Spike (S)) induced inflammatory responses in two macrophages: RAW264.7 and MH-S cells. Macrophages release nitric oxide (NO) and cytokines during LPS-stimulated acute inflammation [31]. This study uses LPS as the positive control. It was firstly determined whether SARS-CoV-2-E and SARS-CoV-2-S induce NO in macrophages and the results show that SARS-CoV-2-E significantly increases the production of NO in a concentration-dependent manner, but not SARS-CoV-2-S (Fig. 1A). Also, SARS-CoV-2-E significantly increases levels of IL-6 (a biomarker that contributes to poor outcomes for patients with COVID-19 [32]) in both macrophages: RAW264.7 and MH-S cells (Fig. 1B). SARS-CoV-2-E also induces TNF-α and IL-12 (Fig. 1B-C). IL-1β and INF-γ are not induced by SARS-CoV-2-E and S (data not shown). These results show that SARS-CoV-2-E has a pivotal role in triggering primary immune responses.

Fig. 1.

SARS-CoV-2-E induces inflammation in macrophages. RAW264.7 and MH-S cells were treated with various concentrations of SARS-CoV-2-E and S (0.1–10 μg/mL) for 24 h. (A) NO production was determined using a Griess assay. (B) The concentrations of IL-6 were determined by ELISA. (C-D) RAW264.7 and MH-S cells were treated with SARS-CoV-2-E and S (1 μg/mL) for 24 h. The concentrations of TNF-α (C), IL-12 (D) were determined by ELISA. The LPS (lipopolysaccharide; 100 ng/mL) treatment group is the positive control. Each E or S-treated group is normalized against the LPS-alone treatment group. The data representatives three independent experiments and is presented as the mean ± SDs; the error bars indicate SD. Significant difference between control (CTL) and SARS-CoV-2-E/S treatment groups is shown as *P < 0.05 and ***P < 0.001.

3.2. GMI inhibits SARS-CoV-2-E-induced inflammatory responses in murine macrophages

GMI is a type of fungal immunomodulatory protein so it is hypothesized that GMI plays a pivotal role in modulating a SARS-CoV-2-induced cytokine storm. Immune or epithelial cells secret inflammatory factors after SARS-CoV-2 infection, which causes tissue damage and a cytokine storm [33]. To determine the role of GMI on inflammation, this study initially determined whether GMI affects the viability of macrophages. Macrophages, RAW264.7 and MH-S cells, are used to determine the cytotoxic effect of GMI. A MTT assay was performed to determine the cell survival rate of RAW264.7 and MH-S cells that are treated with GMI for 24 h. Dexamethasone (DXT), which is a clinical drug for COVID-19 [34], was also used as positive control for anti-inflammation. As shown in Fig. 2A, GMI does not affect the viability of RAW264.7 and MH-S cells at 0.15– 1.2 μM, suggesting that GMI is no cytotoxic for macrophages. However, DXT is significantly cytotoxic to MH-S cells.

Fig. 2.

GMI reduces SARS-CoV-2-E-induced inflammation. (A-B) RAW264.7 and MH-S cells were treated with various concentrations of GMI (0.15–1.2 μM) or DTX (2.5–20 μM) in the absence (A) or presence (B) of SARS-CoV-2-E protein (1 μg/mL) for 24 h. The viability of macrophages was determined by MTT assay. Each GMI-treatment group is normalized against an untreated control. The values represent the means ± SD. Non-significant differences are shown (N.S.) compared with the control group. (C-F) Cells (20,000 cells in 96 well) were stimulated with E proteins (1 μg/mL) in the presence or absence of various concentrations of GMI (0–1.2 μM) or DTX (2.5–20 μM) for 24 h simultaneously. (C) NO production was determined by Griess assay. The concentrations of IL-6 (D), TNF-α (E) and IL-12 (F) were determined by ELISA. The LPS (100 ng/mL) treatment group is the positive control. Each GMI-treated group is normalized against the LPS-alone treatment group. The data represents three independent experiments and is presented as the mean ± SD; the error bars indicate SD. The significant difference between GMI/DXT treatment groups and the control (CTL)/E protein individual treatment group is expressed as *P < 0.05, **P < 0.01, and **P < 0.001.

To determine the effect of GMI on anti-SARS-CoV-2-induced cytokines, SARS-CoV-2-E was used to stimulate macrophages, which served as an in vitro inflammatory system. Initially, co-treatment of GMI and SARS-CoV-2-E does not induce a cytotoxic effect on macrophages (Fig. 2B) but DXT contributes to the cytotoxic effect on SARS-CoV-2-E stimulated MH-S cells. The inflammatory molecules were then examined after macrophages were co-treated with GMI and SARS-CoV-2-E. GMI and DXT significantly inhibit SARS-CoV-2-E-induced NO production in RAW264.7 and MH-S cells (Fig. 2C). The levels of pro-inflammatory cytokines, including IL-6, TNF-α, and IL-12, were also measured in both the macrophages, RAW264.7 and MH-S cells, after co-treatment of GMI and SARS-CoV-2-E. GMI significantly downregulates the secretion of SARS-CoV-2-E-induced cytokines (Fig. 2D-F). In particular, SARS-CoV-2-E induces inflammation in the two types of macrophages. However, GMI has a stronger anti-inflammatory effect on alveolar macrophage MH-S cells than on RAW264.7 cells. The results also show that has a greater anti-inflammatory effect than DXT (Table 1 ). DXT also exhibits excellent anti-inflammatory activity in MH-S cells. These results show that GMI inhibits the cytokine storm that is caused SARS-CoV-2 infection.

Table 1.

EC₅₀ of GMI and DXT in SARS-CoV-2-E-stimulated macrophages.

EC50 (μM)	RAW264.7		MH-S		PMA/THP-1
	GMI	DXT	GMI	DXT	GMI	DXT
NO production	0.70	>20	0.14	<2.5	–	–
IL-6	0.06	>20	0.14	<2.5	1.55	0.56
TNF-α	>1.20	>20	0.85	<2.5	20.45	>80
IL-12	0.18	>20	0.14	<2.5	–	–
Cell viability (IC50)	>1.2	>20	>1.2	>2.5	>1.2	>20.0

3.3. GMI suppresses SARS-CoV-2-E-induced IL-6 and TNF-α in PMA-stimulated THP-1 cells

The effect of GMI on SARS-CoV-2-E-induced human macrophages is also determined. Initially, human monocyte THP-1 cells were stimulated with PMA to create macrophages [35]. The effect of GMI and DXT on PMA-stimulated THP-1 cells were then determined. GMI slightly inhibits the viability of these cells, but not DXT (Fig. 3A). Co-treatment with GMI and SARS-CoV-2-E does not induce a cytotoxic effect on PMA-stimulated THP-1 cells but co-treatment of cells with DXT and SARS-CoV-2-E inhibits viability by 30 % (Fig. 3B). The secretion of cytokine for SARS-CoV-2-E-that are used to treat PMA-stimulated THP-1 cells after GMI treatment was then determined. As shown in Fig. 3C-D, GMI concentration-dependently reduces IL-6 and TNF-α in SARS-CoV-2-E-treated PMA-stimulated THP-1 cells. The concentration-response relationship between GMI and the IL-6 production in human PMA-induced THP-1 cells is consistent with that in mouse macrophage RAW264.7 and MH-S cells. DXT attenuates IL-6 secretion but not TNF-α. GMI better suppresses cytokine than DXT (Table 1). SARS-CoV-2-E does not induce NO production or the secretion of IL-12 on PMA-stimulated THP-1 cells (data not shown). These results show that GMI exhibits anti-inflammatory activity on SARS-CoV-2-E-stimulated human and murine macrophages.

Fig. 3.

GMI inhibits E protein-induced IL-6 and TNF-α in PMA-stimulated human macrophage THP-1 cells. THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) in 100 ng/mL for 24 h, The PMA-induced THP-1 cells (20,000 cells in 96 well) were co-treated with SARS-CoV-2-E protein (1 μg/mL) and various concentration of GMI (0.15–1.2 μM) or DXT (2.5–20 μM) for 24 h. (A-B) The cell viability was determined by a MTT assay. (C-D) The IL-6 and TNF-α protein levels were determined by ELISA methods. The data represents three independent experiments and is presented as the mean ± SD; the error bars indicate SD. The significant difference between GMI/DXT treatment groups and the control (CTL)/SARS-CoV-2-E protein individual treatment group is expressed as *P < 0.05, **P < 0.01, and **P < 0.001.

3.4. GMI downregulates SARS-CoV-2-E-induced multiple inflammatory signaling molecules in macrophages

Similarly to LPS, the results show that SARS-CoV-2-E significantly induces NO production, so SARS-CoV-2-E may regulate intracellular molecules that mediate NO synthesis. Evidence shows that LPS-induced high levels of NO are mediated by inducible nitric oxide synthase (iNOS) [36] so it is therefore hypothesized that SARS-CoV-2-E up-regulates iNOS expression but GMI inhibits the expression of SARS-CoV-2-E stimulation. As shown in Fig. 4A, GMI does not affect iNOS expression but significantly downregulates SARS-CoV-2-E-induced levels of iNOS. In particular, SARS-CoV-2-E does not significantly induce iNOS expression on PMA-stimulated THP-1 cells, which may be why SARS-CoV-2-E does not induce NO production. COX-2 is also the enzyme that is largely responsible for inflammation [37]. SARS-CoV-2-E significantly induces the expression of COX-2 but GMI downregulates COX-2 levels in macrophages that are stimulated with SARS-CoV-2-E (Fig. 4B). Evidence shows that SARS-CoV-2-E induces inflammatory responses via TLR2-mediated NF-κB and ERK pathways [29]. SARS-CoV-2-E does not activate NF-κB (data not shown) but does induce phosphorylation of MAPK pathways, including ERK1/2 and P38. Importantly, GMI eliminates the SARS-CoV-2-E-increased phosphorylation of ERK1/2 and P38 (Fig. 4C). PD98059 (ERK inhibitor) also eliminates SARS-CoV-2-E-induced phosphorylated ERK1/2 and concomitantly reduces the expression of iNOS and COX-2 in murine macrophages, but not SB203580 (P38 inhibitor) (Fig. 4D-F). Interestingly, SB203580 reduces SARS-CoV-2-E-induced expressions of iNOS and COX-2 in PMA-stimulated THP-1 cells (Fig. 4D and F). These results show that GMI inhibits SARS-CoV-2-E-stimulated inflammation via suppression of ERK1/2 and P38.

Fig. 4.

GMI reduces the expressions of SARS-CoV-2-E-induced inflammatory signaling. (A-B) Macrophages (RAW264.7, MH-S and PMA-induced THP-1) were treated with GMI (0.6 μM) for 3 h and stimulated with SARS-CoV-2-E protein (1 μg/mL) for another 24 h. (A) The levels of iNOS. (B) The levels of COX-2. Actin is used as an internal control. (C) Macrophages were co-treated with GMI (0.6 μM) and SARS-CoV-2-E protein (1 μg/mL) for 3 h. (D-F) Macrophages were co-treated with SARS-CoV-2-E protein (1 μg/mL) and PD98059 (ERK inhibitor, 10 μM) or SB203580 (P38 inhibitor, 10 μM) for 3 h (D) and 24 h (E-F). The iNOS, COX-2 and phosphorylated MAPK molecules (ERK, JNK, and P38) were detected using a Western blot assay. Actin and tubulin are used as the internal control. The densitometries of indicated proteins are quantified using Image J software. (G-H) Macrophages were co-treated with SARS-CoV-2-E protein (1 μg/mL) and PD98059 (10 μM) or SB203580 (10 μM) for 24 h. The IL-6 (G) and TNF-α (H) protein levels were determined by ELISA methods. The data represents three independent experiments and is presented as the mean ± SD; the error bars indicate SD. The significant difference between treatment groups and the control SARS-CoV-2-E protein individual treatment group is expressed as *P < 0.05, **P < 0.01, and ***P < 0.001.

3.5. GMI reduces SARS-CoV-2-E-induced pro-inflammatory cytokines in lung tissue of mice

To verify the in vivo anti-inflammatory effect of GMI, an inhalation method was used for the mouse model. The scheme by which the mice received GMI and SARS-Co-V-2-E by the inhalation method is shown in Fig. 5A. The mice were exposed to a SARS-CoV-2-E (20 μg/mL) aerosol that was generated using a nebulizer. Thirty minutes after exposure to the SARS-CoV-2-E, GMI was administered to the mice at 100 μg/mL by exposing the mice to a GMI aerosol that was generated using the nebulizer. The IL-6 production in the lung tissue and blood was measured either 6 h or 24 h after SARS-CoV-2-E administration. However, TNF-α levels in the lung tissue and blood of mice do not change (data not shown). Referring to Fig. 5B-C, the GMI administration following the SARS-CoV-2-E exposure significantly reduces IL-6 production in lung tissue (Fig. 5B) and in blood serum (Fig. 5C) in mice. However, GMI has a less inhibitory effect on IL-6 production 24 h after GMI administration than 6 h after the GMI administration. In addition, the expressions of proinflammatory cytokines in lung tissues were measured. As expected, the results show that SARS-CoV-2-E induces INF-γ, IL-1β, IL-12 and TNF-α, but GMI significantly reduces SARS-CoV-2-E-stimulated inflammatory responses (Fig. 5D-G). These results show that GMI suppresses the cytokine storm that is induced by SARS-CoV-2-E.

Fig. 5.

GMI reduces SARS-CoV-2-E-induced expressions of proinflammatory cytokines in vivo.

(A) The scheme for mice receiving GMI and SARS-CoV-2-E protein by inhalation. Experiments were divided into 6 h and 24 h exposure groups. Mice receiving GMI (100 μg/mL) in the presence or absence of SARS-CoV-2-E (20 μg/mL) by inhalation. After exposure, the mice were sacrificed. The lung tissues and blood serum were harvested. (B-C) The levels of IL-6 in lung tissue (B) and serum (C) were measured by ELISA. (D-G) The levels of IFN-γ (D), IL-1β (E), IL-12 (F) and TNF-α (G) in lung tissue were measured by ELISA. The data represents each experiment and is presented as the mean ± SD; the error bars indicate SD. The significant difference between GMI/ SARS-CoV-2-E protein treatment groups and the control (CTL) group is expressed as *P < 0.05 and **P < 0.001. The significant difference between SARS-CoV-2-E protein treatment groups and the SARS-CoV-2-E + GMI treatment group is expressed as ^#P < 0.05, ^##P < 0.01 and ^##P < 0.001.

4. Discussion

Inflammation is the main cause of a SARS-CoV-2-related cytokine storm and long COVID symptoms [38]. Targeting inflammation is a viable strategy for curing COVID-19. In addition to clinical drugs, natural products are used as a complementary therapy to reduce SARS-CoV-2 infection and to alleviate the COVID-19-related cytokine storm [39]. Previous studies show that GMI alleviates infection of SARS-CoV-2 various mutant Spike-pseudotyped viruses by inducing ACE2 degradation in host cells, inhibiting cell fusion and interfering interaction of spike and ACE2 [22], [23]. This study aims to dissect the effect of GMI on COVID-19-related inflammation using a fast and easy experimental platform. Mouse cells were not able to infect SARS-CoV-2 [40] so this study determines whether viral subunits induce inflammation in macrophages in vitro before there is humanized ACE2 transgenic mouse. The results of the study show that SARS-CoV-2-S is indeed unable to cause the inflammation of mouse macrophages, but SARS-CoV-2-E significantly promotes the inflammation of mouse and human macrophages. These findings are similar to those for previous studies, which demonstrated that SARS-CoV-2-E causes acute respiratory distress syndrome (ARDS)-like pathological damages and inflammatory reactions [29], [41]. Therefore, this study determines the effects of GMI on reducing the inflammation that is induced by SARS-CoV-2-E. The results show that GMI effectively ameliorates SARS-CoV-2-E-induced inflammatory responses in macrophages.

Increasing evidence shows that DXT has a therapeutic effect on patients with severe COVID-19 [34], [42]. However, it does not have a significant effect on patients with mild symptoms. In addition, taking DXT for a long time causes other side effects of immunosuppression. The results of this study show that DXT significantly inhibits the viability of mouse lung macrophages, even though it has a strong anti-inflammatory effect. Importantly, GMI is not toxic to human and murine macrophages and GMI better suppresses SARS-CoV-2-E-induced inflammation. Related studies also show that GMI does not have any toxic reactions [43]. However, the results of this study show that GMI, like DXT [44], temporarily increases blood pressure in GMI-exposed C57BL/6 mice (data not shown). Therefore, patients with hypertension still need to consider whether GMI will cause transient hypertension. The results also show that after mice are exposed to the SARS-CoV-2-E, grooming behavior and home-cage activity decrease but after the administration of GMI, the mice returned to a normal behavior pattern (data not shown). This observation shows that the SARS-CoV-2-E causes inflammation and discomfort in mice. GMI reduces the expression of IL-6 that is induced by SARS-CoV-2-E in the blood and lungs of mice so GMI may restore the behavioral patterns of mice via quick reduction of SARS-CoV-2-E-induced inflammation.

SARS-CoV-2 induces the production of cytokines, in particular IL-6, which may contribute to lung damage and mortality and are considered to be a predictor of disease severity [45]. This study shows that GMI eliminates SARS-CoV-2-E-induced inflammatory markers, especially IL-6 so GMI may directly neutralize inflammatory molecules and address the symptoms of COVID-19-related inflammatory syndrome. However, relevant human trials are required to determine whether GMI reduces the cytokine storm in severe patients. On the other hand, this study establishes a simple cell and animal platform to identify that SARS-CoV-2-E causes a large increase in IL-6 and other cytokines. However, we found that IL-6 in the blood of mice that respond to SARS-CoV-2-E stimulation returns to normal levels after 24 h of exposure (Fig. 5C). Interestingly, when the mice are exposed to GMI for 24 h, IL-6 in the blood increases slightly but the expression of IL-6 is reduced by GMI if there is SARS-CoV-2-E-stimulated inflammation. These results show that GMI inhibits IL-6 in SARS-CoV-2-E-induced inflammation, and GMI slightly activates the immune response in normal mice, which confirms that GMI is an immunomodulatory protein that slight activates an immune response under normal condition, and plays an anti-inflammatory role in excessive immune response. For the limitation, the mechanism by which GMI regulates immunity requires further study. GMI inhibits inflammation if it is inhaled in mouse model, but it is unclear whether this fungal protein also produces an anti-inflammatory effect in the lung environment if it is administered by ingestion or injection.

5. Conclusion

GMI is a fungal immunomodulatory protein (FIP). Currently, >38 FIPs have been identified [18], [19] but very few studies concern the anti-inflammation effect of FIP, especially in terms of the inflammation that is caused by SARS-CoV-2. This study is the first to identify viral subunits that induce inflammation in murine and human macrophages using a simple method. This method quickly screens potential anti-inflammatory drugs/foods without the need for real virus tests or the establishment of humanized ACE2 transgenic mice. The results of the study show that GMI is a potential anti-inflammatory supplement that reduces the inflammatory response that is produced by SARS-CoV-2-E. Future clinical trials should determine the effect of GMI on reducing the cytokine storm in severe patients or in reducing the symptoms of long COVID that are caused by immune abnormalities.

CRediT authorship contribution statement

Z.-H. L., H. Y. and H.-C. L. performed experiments and analyzed data. M.-Y. N., L.-K. W. and T.-T. C. provided conception. W.-J. H. analyzed data. M.-H. Y. provided project administration and funding acquisition. T.-Y. L. provided conception, design of research, analyzed data, wrote and edited the manuscript and funding acquisition. All of authors approved final version of manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.