Solubilized curcuminoid complex prevents extensive immunosuppression through immune restoration and antioxidant activity: Therapeutic potential against SARS-CoV-2 (COVID-19)

The therapeutic benefits of curcuminoids in various diseases have been extensively reported. However, little is known regarding their preventive effects on extensive immunosuppression. We investigated the immunoregulatory effects of a curcuminoid complex (C—S/M), solubilized with stevioside, using a microwave-assisted method in a cyclophosphamide (CTX)-induced immunosuppressive mouse model and identified its new pharmacological benefits. CTX-treated mice showed a decreased number of innate cells, such as dendritic cells (DCs), neutrophils, and natural killer (NK) cells, and adaptive immune cells (CD4 and CD8 T cells) in the spleen. In addition, CTX administration decreased T cell activation, especially that of Th1 and CD8 T cells, whereas it increased Th2 and regulatory T (Treg) cell activations. Pre-exposure of C—S/M to CTX-induced immunosuppressed mice restored the number of innate cells (DCs, neutrophils, and NK cells) and increased their activity (including the activity of macrophages). Exposure to C—S/M also led to the superior restoration of T cell numbers, including Th1, activated CD8 T cells, and multifunctional T cells, suppressed by CTX, along with a decrease in Th2 and Treg cells. Furthermore, CTX-injected mice pre-exposed to C—S/M were accompanied by an increase in the levels of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase), which play an essential role against oxidative stress. Importantly, C—S/M treatment significantly reduced viral loads in severe acute respiratory syndrome coronavirus 2-infected hamsters and attenuated the gross pathology in the lungs. These results provide new insights into the immunological properties of C—S/M in preventing extensive immunosuppression and offer new therapeutic opportunities against various cancers and infectious diseases caused by viruses and intracellular bacteria.


Introduction
Curcuminoids are the major polyphenolic compounds found in the rhizomes of turmeric (Curcuma longa), which belong to the Zingiberaceae family. It is one of the few prospective natural compounds that has been extensively studied from a pharmacological perspective [1,2]. The main components of curcuminoids are known to be curcumin (~77 %), demethoxycurcumin (~17 %), and bisdemethoxycurcumin (~3%) [3]. In the history of clinical studies, curcuminoids have been verified to have excellent safety and drug tolerance, approved by the Food and Drug Administration (FDA) as "Generally Recognized as Safe (GRAS)," which are applicable in the form of food, drugs, and cosmetics without legal restrictions [4,5]. Based on these advantages, the biological activities, such as antiviral, anti-inflammatory, anticoagulant, antiplatelet, antioxidant, and immunomodulatory properties, of curcuminoids have been extensively studied. Among them, an anti-inflammatory effect has been reported as a significant feature of curcuminoids [6][7][8]. The antiinflammatory functions of curcuminoids result from their ability to inhibit signals (such as mitogen-activated protein kinases [MAPKs], nuclear factor kappa-B [NF-κB], and Janus kinase [JAK]/signal transducer and activator of transcription [STAT]-dependent signals) of widespread inflammation, thereby promoting their protective efficacy against various inflammatory diseases [9]. However, despite their wide range of potential benefits, their poor bioavailability and low solubility have hindered their clinical application [10]. Therefore, resolving these disadvantages will not only promote the clinical use of curcuminoids but also provide an opportunity to find new indications by discovering new biological functions.
During the development of therapeutic agents and enhancers for various diseases, a wide spectrum of drug delivery systems has been reported to improve the low bioavailability and solubility of candidate compounds. One technique is micellar solubilization, in which hydrophobic drugs are solubilized in the inner core, on the surface, or the palisade layer of micelles [11]. The second technique is nanosizing, in which the size of drug particles is altered to the nanometer level (≤1 μm) using high-pressure homogenization [12]. The third technique is nanoencapsulation drug delivery system, in which drugs are enclosed in plant-derived eco-friendly liposomal or extracellular vesicles similar to liposomes for effective delivery to target cells [13,14]. All these techniques not only improve the solubility of drugs but also lower their instability against various in vivo risk factors (such as physiological temperature and pH changes) [15,16]. In conclusion, a drug delivery system is widely used to compensate for various issues (including the low bioavailability and solubility) of drugs, and these techniques have been widely adopted to increase the bioavailability of curcuminoids [17,18].
Following this trend, we recently reported a new method for increasing the solubility of curcuminoids using stevioside, a known natural solubilizer, and a microwave-assisted method. Here, watersoluble curcuminoids (named C-S/M) prepared using stevioside are made in nanoparticle form (average particle size of approximately 190 nm) [19]. Importantly, recent studies demonstrated that using biomaterials encapsulated in stevioside can usually improve bioactivity, stability, and cell-to-nanoparticle interaction [20,21]. These results led us to confirm the changed or enhanced biological functions of C -S/M. Cyclophosphamide (CTX) is known to profoundly affect host immune systems as a pharmacological suppressor of innate and adaptive immune responses [22,23]. In particular, CTX has been shown to promote immunosuppressive effects by decreasing the systemic activation of Th1 immunity that promotes protective action against cancers and intracellular pathogens [24]. Thus, the CTX-induced immunosuppression model may help in developing treatments for intracellular pathogen-induced malignancies and infectious diseases.
In this study, to elucidate the immunological advantages of C-S/M nanoparticles and their novel biological functions, we investigated the pharmacological characteristics of C-S/M in CTX-induced extensive immunosuppression. Additionally, we evaluated the therapeutic effect of C-S/M against viral infection based on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected hamsters.

Preparation of water-soluble curcuminoid complex
The curcuminoid (Sigma-Aldrich, St. Louis, MO, USA) and stevioside (1.968 mg; Sigma-Aldrich) mixture (curcuminoids/stevioside mixture) used in the preparation of water-soluble curcuminoids were dissolved in 200 μL of deionized water (DW). The curcuminoids/stevioside mixture (200 μL), curcuminoids (0.032 mg mixed with 200 μL of DW), and stevioside (1.968 mg mixed with 200 μL of DW) were exposed to microwave irradiation using a microwave-assisted extractor (CEM, Matthews, NC, USA) at 100 W and 25 • C for 5 min. The samples were stored at 4 • C until further use. Water-soluble curcuminoids prepared using stevioside and a microwave-assisted method were named C -S/M.

Immunosuppressive mouse model induced by CTX treatment
Female C57BL/6 mice (weighed approximately 20 g, 7-week-old, five mice per group) used in CTX-induced immunosuppression models were purchased from OrientBio Inc. (Seongnam, South Korea) and acclimatized for 7 days to pathogen-free conditions prior to CTX treatment. The mice were housed in pathogen-free rooms at the Korea Research Institute of Bioscience and Biotechnology (KRIBB) animal facility maintained at 22 ± 2 • C, light/dark cycle of 12-h/12-h, and humidity of 55 ± 5 %. The protocols for the CTX-induced immunosuppression mouse model were reviewed and approved by the animal ethics committee of KRIBB (permit number: KRIBB-AEC-21330). For the control groups (Group 1; G1), mice (n = 5 mice) were orally administrated with 200 μL of DW for 5 consecutive days. Three days after DW administration, 200 μL of DW was injected into the mice via the intraperitoneal route for 5 consecutive days and then sacrificed after 6 days. For the CTX treatment groups (Group 2; G2), mice (n = 5 mice) were orally administrated with 200 μL of DW for 5 consecutive days. Three days after DW administration, 100 μL of CTX (80 mg/kg; Sigma-Aldrich) was intraperitoneally administered to the mice for 5 consecutive days, and then the mice were sacrificed after 6 days. For the C-S/M (Group 3; G3), curcuminoid (Group 4; G4), and stevioside (Group 5; G5) treatment groups, mice (n = 5 mice) were orally administrated with 200 μL of DW containing C -S/M (100 mg/kg), curcuminoids (1.6 mg/kg), and stevioside (98.4 mg/kg) for 5 consecutive days, and then the mice were injected with 100 μL of CTX (80 mg/kg) intraperitoneally for 5 consecutive days. G3, G4, and G5 mice were sacrificed after 6 days. Spleen and blood samples were collected from sacrificed mice in all groups. Body weights were analyzed at 0, 6, and 17 days.

Flow cytometry analysis for absolute number and activation of natural killer cells
Single-cell suspensions resuspended in the cRPMI medium were seeded into U-shaped bottom microplates (Corning, New York, NY, USA) and incubated in the absence or presence of 1 × Cell Stimulation Cocktail (plus protein transport inhibitors; Invitrogen) containing phorbol 12-myristate 13-acetate and ionomycin for 4 h at 37 • C. The cells were harvested and stained with L/D, lineage antibody cocktail, and anti-NK1.1 mAb for 30 min at 4 • C. After surface staining, the cells were washed with cold PBS and pelleted by centrifugation (at 1500 × g for 3 min at 4 • C). The pelleted cells were resuspended with 100 μL of Cytofix/Cytopermsolution (BD Bioscience) per well for 30 min at 4 • C. Finally, the cells were washed with 1 × Perm/Wash solution and stained with anti-CD107a, anti-IFN-γ, and anti-granzyme B mAbs. Absolute numbers (in the non-treatment condition of Cell Stimulation Cocktail; the number of cells per 100 μL of 10 mL of the total single cell suspension) and activation (in the absence and presence of Cell Stimulation Cocktail) of NK cells were analyzed using a flow cytometer and the FlowJo software.

Flow cytometry analysis for absolute number and activation of neutrophils
Single-cell suspensions were stained with L/D, lineage antibody cocktails, and anti-Gr-1, anti-CD11b, and anti-CD62L mAbs for 30 min at 4 • C. After PBS washing, absolute numbers (the number of cells per 100 μL of 10 mL of the total single cell suspension) and activation of neutrophils were analyzed using a flow cytometer and thr FlowJo software.

Flow cytometry analysis for the absolute number of CD4 + and CD8 + T cells
Single-cell suspensions were stained with L/D, anti-CD3, anti-CD4, and anti-CD8 mAbs for 30 min at 4 • C in the dark, washed with cold PBS, and pelleted by centrifugation. The absolute numbers (the number of cells per 100 μL in 10 mL of the total single cell suspension) of CD3 + CD4 + and CD3 + CD8 + T cells were analyzed using a flow cytometer and the FlowJo software.

Flow cytometry analysis of Th1, Th2, Th17, Treg, and multifunctional T cells
Single-cell suspensions were seeded into U-shaped bottom microplates and incubated in the absence or presence of 1 × Cell Stimulation Cocktail for 4 h at 37 • C. After stimulation, the cell surfaces were stained with L/D, anti-CD3, anti-CD4, anti-CD8, and/or anti-CD25 mAbs for 30 min at 4 • C. The cells were then washed with cold PBS and centrifuged at 1500 × g for 3 min at 4 • C. The pelleted cells were resuspended in 100 μL of a Cytofix/Cytoperm solution (for Th1, Th2, Th17, multifunctional T cell analysis; BD Bioscience) or transcription factor staining buffer (for Treg cell analysis; ThermoFisher Scientific) per well for 30 min at 4 • C, and the cells were washed with 1 × Perm/Wash solution. Next, the cells were stained with antibody cocktails for Th1/Th2/Th17 cell detection (anti-IFN-γ, anti-IL-5, and anti-IL-17A mAbs), multifunctional T cell detection (anti-IFN-γ, anti-IL-2, and anti-TNF-α mAbs), or Treg cell detection (Foxp3 mAb) for 30 min at 4 • C. After intracellular staining, T cells were analyzed using a flow cytometer and the FlowJo software.

Analysis of antioxidant enzymes
Serum samples from normal and CTX-induced immunosuppressed mice were diluted (2 × ) in PBS, and antioxidant enzymes were analyzed using the superoxide dismutase (SOD) activity kit (Invitrogen), catalase activity kit (Invitrogen), and glutathione peroxidase kit (Abcam, Cambridge, MA, USA) according to the manufacturer's protocol.

In vivo antiviral activity against SARS-CoV-2
To investigate the gross lung lesion and virus replication in response to SARS-CoV-2 infection and C -S/M treatment, female WT Golden Syrian hamsters (7-week-old) were purchased from Janvier Labs (Janvier Labs, Saint-Berthevin, France) and acclimatized for 7 days to pathogen-free conditions. The mice were housed in a BL-3 biohazard animal facility of the KRIBB and maintained at 22 ± 2 • C, light/dark cycle of 12-h/12-h, and humidity of 40-55 %. In the animal experiments, all procedures were reviewed and approved by the animal ethics committee of KRIBB (permit number: KRIBB-AEC-21329 and KRIBB-IBC-20220201). The care and handling of animals complied with all of the current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85- 23, 1985, revised 1996). The strain of SARS-CoV-2, KCDC03 (isolated from a Korean COVID-19 patient in 2020 and belonging to the A lineage of early Chinese strains), was kindly provided by the Korea Disease Control and Prevention Agency (Cheongju, South Korea) and propagated in Vero E6 cells (ATCC, Manassas, VA, USA) as previously described [25]. For therapeutic effects of C-S/M against SARS-CoV-2-infected hamsters, virus alone-infected and C -S/M− treated groups (n = 5/groups) were challenged once by intratracheal inoculation of 10 5 TCID 50 of SARS-CoV-2 in 100 μL PBS. After the infection had passed, the C -S/ M− treated groups were administered orally with C-S/M (218.75 mg/ head) each in 100 μL of DW with a 12 h interval for four and a half consecutive days. Whole lung tissues were sampled from hamsters in each group at 5 days post-infection, and the gross lung lesion surfaces were examined using the ImageJ software (National Institutes of Health, United States) after taking photographs of the dorsal and ventral lung surfaces. Also, virus growth in the lungs of the hamsters was evaluated as previously described [25]. Briefly, total RNA was extracted using the RNeasy kit (Qiagen, Hilden, Germany) in homogenized lung tissues, and cDNA was synthesized using the TOPscript™ cDNA Synthesis kit (Enzynomics, Daejeon, South Korea) according to the manufacturer's instructions. First-strand cDNA samples were subjected to quantitative real-time PCR using TOPreal™ qPCR 2X PreMix (Enzynomics), and CFX96 qPCR cycler (Bio-Rad Laboratories, Hercules, California, USA) was applied with the following conditions using SARS-CoV-2 N-specific primer (Forward 5 ′ -TAA TCA GAC AAG GAA CTG ATT A-3 ′ ; Reverse: 5 ′ -CGA AGG TGT GAC TTC CAT G-3 ′ ). The reduction in viral load was expressed as relative values, where genome copy numbers in virusinfected and C-S/M− treated groups were normalized to those in virus-infected controls.

Statistical analyses
Statistical analyses of the data were performed using the GraphPad statistical software (San Diego, CA, USA). Comparison between two groups was performed using an unpaired Student's t-test. Comparison between more than two groups was made using one-way ANOVA with Tukey's multiple comparison test, where a *p < 0.05 was considered statistically significant.

C-S/M pre-treatment alleviates a decrease in immune cell populations and body weight loss observed in CTX-induced immunosuppressed mice
CTX is a chemotherapeutic drug indicated for some cancers and autoimmune diseases. It leads to extensive immunosuppression in the host immune system [22,23]. In this study, we investigated whether C -S/M could prevent widespread immunosuppression. Animal experimental methods are shown in Fig. 1A. CTX alone-injected mice (G2) showed a significant decrease in body weight compared with that of the G1 group. Interestingly, the mouse body weight loss induced by CTX injection was recovered by pre-exposure to C-S/M (G3); however, this weight change was not significantly different in curcuminoid (G4)-and stevioside (G5)-administered mice compared with that of the G2 group (Fig. 1B). In addition, CTX injection (G2) showed a remarkable decrease in innate (particularly DCs, neutrophils, and NK cells) and adaptive (CD4 + and CD8 + T cells) immune cell numbers in the spleens of mice compared with those in the G1 group, but this decrease was restored in CTX-injected mice pre-exposed to C -S/M (G3). In contrast, monotherapy with curcuminoids (G4) and stevioside (G5) was ineffective in preventing immune cell loss in CTX-injected mice. Unlike other innate cells, increased numbers of macrophage were observed in CTX-injected mice (G2) than in the DW (G1)-and C -S/M (G3)-injected groups ( Fig. 1C and D). The results induced by C-S/M pre-treatment led us to investigate further the functional recovery effect of the innate (particularly DCs, neutrophils, and NK cells) and adaptive cell responses in immunosuppressed mice.

C-S/M pre-treatment restores suppressed the splenic DC maturation and macrophage activation observed in CTX-induced immunosuppressed mice
We focused on the activation of antigen-presenting cells (APCs, including DCs and macrophages), as these cells are important for activating adaptive immunity and contributing to immunogenic tolerance [26,27]. Therefore, when flow cytometry was performed to analyze splenic DC maturation ( Fig. 2A) and macrophage activation (Fig. 2C), mice exposed to CTX alone (G2) showed lower expression of surface molecules (CD80, MHC-I, and MHC-II) on lineage -CD11c + splenic DCs and lineage -F4/80 + macrophages than the G1 group. Interestingly, CTXinjected mice pre-exposed to C -S/M (G3) restored CD80 and MHC-II expression in splenic DCs but not MHC-I expression levels ( Fig. 2A and  B). Additionally, CTX-injected mice pre-exposed to C -S/M (G3) restored CD80, MHC-I, and MHC-II expression in macrophages ( Fig. 2C  and D). Importantly, oral administration of curcuminoids (G4) and stevioside (G5) did not restore the reduced surface molecule expression in splenic DCs and macrophages observed in CTX alone (G2)-injected mice. These results indicate that C-S/M restores the APC activation inhibited by CTX and might prevent the reduced CTX-mediated T-cell response because the expression of CD80, MHC-I, and MHC-II on APCs is closely related to the activation, proliferation, and differentiation of CD4 + and CD8 + T cells when interacting with APCs and T cells [28].

C-S/M pre-treatment increases functional restoration of NK cell activation reduced in CTX-induced immunosuppressed mice
NK cells that secrete various cytokines (TNF-α and IFN-γ) and cytotoxic molecules (granzyme B and perforin) play important roles in controlling and eliminating unwanted cells, such as cancer and virusand bacteria-infected cells [29,30]. These cells can also promote the activity of APCs and T cells [31]. Therefore, we investigated whether C -S/M treatment could prevent the decrease in NK cell activation induced by CTX administration in mice. To accomplish this, single-cell suspensions from the spleen were stimulated with Cell Stimulation Cocktail, and the expression of CD107a (a marker of cytotoxic NK cells), IFN-γ, and granzyme B as markers of NK cell (lineage -NK1.1 + cells) functional activity was analyzed using flow cytometry (Fig. 3A). Similar to the ability of CTX to inhibit APC activation, CTX also inhibits NK cell activation (NK cells expressing CD107 + granzyme B + IFN-γ + , CD107 + IFN-γ + , CD107 + granzyme B + , CD107 + , and IFN-γ + ). Importantly, C-S/M− administered CTX-injected mice (G3) showed a higher frequency of CD107 + granzyme B + IFN-γ + -expressing NK cells than the G2 group but not the G4 and G5 groups ( Fig. 3B and C).

C-S/M pre-treatment increases functional restoration of neutrophil activation reduced in CTX-induced immunosuppressed mice
Neutrophils, which are important innate cells involved in the clearance of acute bacterial and viral infections, may help activate APCs, NK cells, and T cells [32,33]. To evaluate whether neutrophil activation is increased or decreased by C-S/M treatment, we next assessed neutrophil activation in CTX-injected mice following pre-treatment with C -S/ M. When flow cytometry was performed to assess neutrophil activation in the spleens of each mouse (Fig. 4A and B), mice exposed to CTX alone (G2) showed lower frequencies of activated neutrophils (lineage -CD11b + Gr-1 + CD62Lcells) than the G1 group. Here, CD11b and Gr-1 are specific markers for neutrophils. CD62L levels decrease when neutrophils are activated [34]. Interestingly, CTX injection in mice preexposed to C -S/M (G3) restored neutrophil activation but not in the G4 and G5 groups. Based on the above results, we predicted that the recovery effects of the activation of APCs, NK cells, and neutrophils mediated by C -S/M could contribute to restoring CTX-induced T-cell activities.

C-S/M pre-treatment encourages the immuno-enhancing effects for Th1 and CD8 + T cells in CTX-induced immunosuppressed mice
To validate our hypothesis for T cells, an additional analysis was performed that was focused on subtypes (Th1, Th2, Th17, and Treg cells) and/or activation of CD4 + and CD8 + T cells induced by C -S/M pre-treatment in CTX-injected mice. We found that the CTX-aloneinjected group (G2) had significantly decreased frequencies of IFNγ + CD3 + CD4 + (Th1) and IFN-γ + CD3 + CD8 + T cells (activated CD8 T cells or cytotoxic T lymphocytes; CTL) compared with those of the control group (G1), while the frequencies of IL-5 + CD3 + CD4 + (Th2) and Foxp3 + CD25 + CD3 + CD4 + (Treg) cells were increased. Th17 cell frequencies did not differ between the groups. These results demonstrated that immunosuppressive responses of CTX could be achieved through a decrease in Th1 and activated CD8 + T cells (CTL) and an increase in Th2 and Treg cells. Herein, Th1 and activated CD8 + T cells can induce host defense mechanisms against cancers, viral infections, and intracellular bacterial infections [35,36]. In contrast, Th2 cells can inhibit the activation of Th1 and CD8 + T cell responses, and Treg cells can suppress the activation of various immune cell types, including B cells, NK cells, and pan-T cells [37][38][39]. Interestingly, C-S/M− administered CTX-injected mice (G3) showed significantly increased frequencies of Th1 and activated CD8 + T cells compared to that of the G2 group; further, these mice were accompanied by a decrease in Th2 and Treg cells (Fig. 5A and B). These data indicate that C-S/M can restore Th1 and activated CD8 + T cell responses with a decrease in Th2 and Treg cells in a wide range of immunosuppressive actions.

C-S/M pre-treatment promotes multifunctional T-cell responses in CTX-induced immunosuppressed mice
To further investigate the resilience of Th1 and CTL responses induced by C-S/M, we confirmed whether C-S/M can induce multifunctional T cells capable of producing multiple Th1 cytokines, which are functionally superior to Th1 and CD8 + T cells. To achieve this, we analyzed multifunctional CD4 + and CD8 + T cells capable of producing multiple Th1 cytokines (IFN-γ, TNF-α, and IL-2) in the spleen of each group by multicolor flow cytometric analysis (Fig. 6A). CTX aloneinjected mice (G2) presented with a remarkable decrease in the frequency (Fig. 6B) and proportion (Fig. 6C) of multifunctional CD4 + (CD3 + CD4 + cells co-expressing IFN-γ + TNF-α + IL-2 + ) and CD8 + (CD3 + CD8 + cells co-expressing IFN-γ + TNF-α + IL-2 + ) T cells than those of normal mice (G1). Importantly, the reduction in multifunctional CD4 + and CD8 + T cells in CTX alone-injected mice was recovered only by C-S/M pre-treatment ( Fig. 6B and C). These results strongly suggest that C-S/M has a strong functionality to restore multifunctional CD4 + and CD8 + T cell responses against extensive immunosuppression.

C-S/M pre-treatment increases antioxidant enzymes reduced in CTXinduced immunosuppressive mice
CTX can cause adverse side effects, such as immunotoxic and immunosuppressive effects, in the host [40]. Thus, we hypothesized that if extensive immunosuppressive action (a decrease in innate and adaptive immune cell activation) induced by CTX occurred through the reduction of the host's antioxidant activity, the immune recovery effects were because of increased antioxidant activity induced due to preexposure of C-S/M in CTX-injected mice. Fig. 7 shows the antioxidant enzyme levels in the serum of each group, CTX-injected mice (G2) had significantly lower levels of antioxidant enzymes (SOD, catalase, and glutathione peroxidase) than the control mice (G1). In contrast, antioxidant activity (SOD, catalase, and glutathione peroxidase) was Fig. 6. Effects of C-S/M pre-administration on multifunctional T cell responses in a CTX-induced immunosuppressive mouse model. At 6 days after the final CTX injection, single-cell suspensions isolated from spleens of each group were incubated with Cell Stimulation Cocktail (plus protein transport inhibitors). Following incubation for 4 h, cells were immunostained for L/D viability dye, anti-CD3, anti-CD4, anti-CD8, anti-IFN-γ, anti-TNF-α, and anti-IL-2 mAbs using an intracellular staining protocol, as described in the material and methods section. (A) Flow cytometry gating strategy for multifunctional CD4 + (L/D -CD3 + CD4 + cells co-producing IFN-γ, TNF-α, and/or IL-2) and CD8 + (L/D -CD3 + CD8 + cells co-producing IFN-γ, TNF-α, and/or IL-2) T cells. (B) Bar graphs show the means ± SD (n = 5 mice per group) of multifunctional CD4 + and CD8 + T cells. (C) The pie charts show the mean (±SD, n = 5 mice per group) frequencies of CD4 + and CD8 + T cells co-expressing IFN-γ, TNF-α, and/or IL-2. Data shown as one representative plot out of three independent experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001. significantly enhanced in CTX-injected mice pre-exposed to C -S/M (G3; p < 0.001 for SOD, catalase, and glutathione peroxidase) and curcuminoids (G4; p < 0.02 for SOD, p < 0.01 for catalase, p < 0.03 for glutathione peroxidase) compared with that in the G2 group but not in the G5 group. These results suggest that the strong antioxidant activity induced by C-S/M may help restore immune cell activity in CTX-induced immunosuppression models.

C-S/M reduces the viral replication and the gross pathology in the lungs of SARS-CoV-2-infected hamsters
Based on the results of improved immunological responses and antioxidant activity induced by C -S/M, we evaluated the protective efficacy of C -S/M in SARS-CoV-2-infected hamsters. Since the activation of adaptive immune responses (particularly CD4 + and CD8 + T cell responses) and an increase in antioxidant activity are drawing attention as therapeutic strategies for SARS-CoV-2 infection [41,42], we chose to use this animal model. At 5 days post-infection, SARS-CoV-2-infected hamsters exposed to C-S/M exhibited attenuation of the gross pathology (Fig. 8A) in the lungs compared to that in the SARS-CoV-2 aloneinfected control groups. In addition, C-S/M− treated groups (p < 0.04) significantly reduced viral loads (Fig. 8B) in the lungs of SARS-CoV-2infected hamsters, indicating that C-S/M can be used as a potential candidate for inducing antiviral activity.

Discussion
Curcuminoids have a broad spectrum of biological functions. Along with major functionality such as anti-inflammatory action, it mediates several immunological and antioxidant processes like restoration of immune cell populations (such as innate and adaptive immune cells) and suppression of immune cell death [8,43]. However, these biological activities are limited by various problems, including the low bioavailability and solubility of curcuminoids. To solve these issues and derive new immunological functions, our study investigated the pharmacological characteristics of solubilized curcuminoids (C-S/M) against the extensive immunosuppression induced by CTX. The notable pharmacological characteristics of C-S/M are discussed ahead. First, preexposure to C -S/M prevented the decrease in innate (particularly DCs, neutrophils, and NK cells) and adaptive immune cell numbers induced by CTX administration in mice. Second, C -S/M showed a preventive function against the suppression of splenic DCs, macrophage, NK cells, neutrophils, Th1, and CD8 + T cell (CTL) activities induced by CTX. Specifically, our study verified the functionality of C-S/M in preventing a decrease in multifunctional Th1 and CD8 + T cells, indicating stronger Th1 and CD8 + T cell responses. Third, the activation of Th2 cells (cells inducing humoral responses and suppressing the activation of Th1 cells) and Treg cells (cells suppressing the activation and proliferation of T cells) increased by CTX could be suppressed by the preadministration of C-S/M. These effects were not observed when curcuminoids and stevioside, the two major components of C -S/M, were used independently. Finally, we showed that C-S/M can attenuate the viral replication and the gross pathology in the lungs of SARS-CoV-2infected hamsters. These results demonstrate the potential of C-S/M in preventing extensive immunosuppression while increasing the antiviral activity against pathogenic viruses. Fig. 7. Effects of C -S/M pre-administration on antioxidant activity in a CTX-induced immunosuppressive mouse model. At 6 days after the final CTX injection, serum samples (n = 5 mice per group) of each group were analyzed for antioxidant activity using the superoxide dismutase (SOD) activity kit, catalase activity kit, and glutathione peroxidase kit. Bar graphs show the means ± SD of antioxidant enzymes. Data shown as one representative plot out of three independent experiments are shown. *p < 0.05, ***p < 0.001. Th1 and CD8 + T cell activation is widely known as a host defense mechanism against cancers, viral infections, and intracellular bacterial infections, and it is used as an immunity marker critical for developing therapeutic agents and vaccines against these pathogens [35,36,44,45]. Th1 and CD8 + T cell activation, differentiation, and proliferation can be induced by interactions between naïve T cells and innate immune cells, especially APCs, including DC and macrophages [46]. Furthermore, cytokines and cell receptors induced by the activation of APCs and T cells can promote the activation of NK cells, both directly and indirectly [47]. NK cells can then increase the activation, proliferation, and differentiation of APCs and T cells [31]. Although determining which immune cell activation was preferentially restored in the process of C -S/ M− mediated prevention of the decreased activation of various immune cells induced by CTX treatment is difficult, our study results indicate that C-S/M could be a candidate compound with the potential to prevent extensive immunosuppression.
Furthermore, materials that can induce the activation of multifunctional T cells can be classified as potential candidate compounds for establishing a strong host immune system against pathogens in the development of therapeutics [48,49]. Multifunctional CD4 + and CD8 + T cells can induce Th1 cytokines, especially IFN-γ, IL-2, and TNF-α, and demonstrate higher cytokine production than cells that induce only one type of cytokine, consistent effector function, and induction of effective expansion of memory T cells [48,50]. These cells can induce highly effective destruction of pathogens by establishing a strong host defense immune system [48,50,51]. From this perspective, C -S/M can prevent the decrease in multifunctional Th1 and CD8 + T cells induced by CTX and contributes to the re-establishment of an effective immune system against various pathogens that suppresses multifunctional T cells.
The production of excessive reactive oxygen in the host and reduced potential of inducing antioxidant effects can cause various challenges, such as suppression of immune cell activation, survival, proliferation, and differentiation [52][53][54]. For example, direct and indirect oxidative stress mediators induced in the tumor microenvironment can promote cancer cell growth by inhibiting the function of effector T cells and inducing T cell death [55,56]. Such immune cell abnormalities due to oxidative stress can be observed in tuberculosis, a major intracellular bacterial infection [52]. Tubercular bacilli can cause persistent and excessive infection of host immune cells. The infected innate immune cells, especially APCs, can promote cellular hypofunction or death by inducing uncontrolled oxidative stress [53,57]. Ultimately, these immune cells lose their defense mechanism against tubercular bacilli, resulting in delayed or suppressed T cell activation, which is a necessary element in host defense immunity [57]. This can also occur in various viral infections, such as SARS-CoV-2 infection and human immunodeficiency virus (HIV) dementia [41,58,59]. Therefore, an excessive amount of reactive oxygen species mediated by various pathogens can be a target for enhancers or therapeutic agents of diseases [55,56]. Cyclophosphamide can induce an excessive amount of reactive oxygen species and ultimately lead to cell damage and extensive immunosuppression [24,60,61]. Pre-treatment with C -S/M restored the levels of antioxidant enzymes reduced by CTX, which further restored the activation and number of immune cells. Although not observed in treatment with curcuminoids or stevioside independently, the preventive effect of C -S/M against extensive immunosuppression is likely because of the improved antioxidative activity resulting from the increased solubility of curcuminoids. These results (improved antioxidant and immune cell activities) led us to investigate the therapeutic activity of C-S/M against SARS-CoV-2 infection. Nevertheless, to derive a more specific therapeutic mechanism for C-S/M, further studies are needed to confirm the immunological responses and antioxidative activities in a SARS-CoV-2-infected animal model exposed to C -S/M.
In summary, our study elucidated the immunological advantages of C -S/M, with improved solubility of curcuminoids, as a preventive agent against extensive immunosuppression. Our results showed that C -S/M can prevent innate and adaptive immunosuppression induced by CTX and increase the levels of various antioxidant enzymes. It is worth noting that C -S/M can induce the activation of suppressed adaptive immune cells through Th1-and CD8 + T cell-dominated responses. In conclusion, we propose the potential of C -S/M as a candidate compound for strengthening various host defense systems that can be suppressed by a wide range of pathogens (viruses and intracellular bacteria) and cancers.

Data availability
Data will be made available on request.

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.

Data availability
Data will be made available on request.