Natural antiviral compound silvestrol modulates human monocyte‐derived macrophages and dendritic cells

Abstract Outbreaks of infections with viruses like Sars‐CoV‐2, Ebola virus and Zika virus lead to major global health and economic problems because of limited treatment options. Therefore, new antiviral drug candidates are urgently needed. The promising new antiviral drug candidate silvestrol effectively inhibited replication of Corona‐, Ebola‐, Zika‐, Picorna‐, Hepatis E and Chikungunya viruses. Besides a direct impact on pathogens, modulation of the host immune system provides an additional facet to antiviral drug development because suitable immune modulation can boost innate defence mechanisms against the pathogens. In the present study, silvestrol down‐regulated several pro‐ and anti‐inflammatory cytokines (IL‐6, IL‐8, IL‐10, CCL2, CCL18) and increased TNF‐α during differentiation and activation of M1‐macrophages, suggesting that the effects of silvestrol might cancel each other out. However, silvestrol amplified the anti‐inflammatory potential of M2‐macrophages by increasing expression of anti‐inflammatory surface markers CD206, TREM2 and reducing release of pro‐inflammatory IL‐8 and CCL2. The differentiation of dendritic cells in the presence of silvestrol is characterized by down‐regulation of several surface markers and cytokines indicating that differentiation is impaired by silvestrol. In conclusion, silvestrol influences the inflammatory status of immune cells depending on the cell type and activation status.

decreased risk of escape mutations by the virus, 8 but also presents difficulties compared to viral targets, such as possible pleiotropic side effects. 9 However, the inhibition of eIF4A by silvestrol appears to be highly specific which should minimize the risk of side effects.
Silvestrol showed, moreover, a broad range of potent antiviral effects on different RNA viruses. For instance, silvestrol inhibits the replication of Coronaviruses, 10 Ebola virus, 11 Zika virus 12 as well as subtypes of Picornaviruses, 10 Chikungunya virus 13 and reduces the release of hepatitis E virus infectious particles. 14 Some intracellular bacterial pathogens have developed sophisticated strategies to prevent M1-like polarization of macrophages, thereby altering microbicidal mechanisms or driving the polarization towards an M2 phenotype to reduce the defensive host inflammatory response. 15 In this respect, it is noteworthy that several antibiotics are able to activate the host immune system and thereby increase immune defence mechanisms independently of the direct drug impact on the microorganism. 16 Such modulation of the immune system can broaden the drug efficacy profile boosting innate host defence mechanisms and thereby increasing pathogen clearance while reducing unwanted tissue damage by extenuated inflammation. Because silvestrol regulates the translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor 17 that promotes innate and adaptive immune responses, 18 we speculated that silvestrol possibly interacts with the host immune system and thereby bolsters its antipathogenic effect and/or promotes resolution of inflammation and tissue damage.

Most infectious diseases are accompanied by local inflammation
and accumulation of various immune cells, such as monocytes, macrophages and dendritic cells, at the site of infection, where they release a broad range of cytokines, chemokines and lipid mediators, which facilitate pathogen clearance. To minimize the tissue damage resulting from exaggerated inflammation, well-timed resolution is essential.
Macrophages play a major role in initiation and resolution of inflammation. They initiate the local inflammation through release of cytokines such as interleukin (IL)-1β, interferon (IFN)-γ, IL-23 and tumour necrosis factor (TNF)-α and recruit further pro-inflammatory immune cells by the release of chemokines (eg CC-chemokine ligand (CCL)2, C-X-C motif chemokine (CXCL)10, IL-8). 19 Macrophages and dendritic cells recognize microbial carbohydrates and mediate phagocytosis via pattern recognition receptors such as CD206 or CD209. 20,21 Thereby, macrophages ingest invading pathogens and present pathogenic peptides via HLA-DR to T cells for the activation of the acquired immune system. M2 macrophages also release cytokines such as IL-10 to support the process of tissue healing and remodelling 22 and chemokines such as CCL18 or CCL17 to recruit anti-inflammatory T H 2 and T reg cells. 19,23,24 Dendritic cells are mainly responsible for the presentation of antigens, the control of the antigen-specific response of T cells and the intensity of the inflammatory process. Activation of dendritic cells induces their expression of co-stimulation molecules (eg CD80, CD86) and HLA-DR.
In the present study, we investigated the immunomodulatory effects of the natural compound silvestrol on human monocyte-derived macrophages (MdMs) and dendritic cells (MdDCs). For this purpose, we isolated CD14 + cells from fresh human blood samples and examined the impact of silvestrol on cell viability, cell-type-specific surface markers, released cytokines and energy metabolism during differentiation and polarization.

| Cells and reagents
Human monocytes, macrophages and dendritic cells were cultured in RPMI1640-Glutamax medium supplemented with 1% penicillin/streptomycin 10% FCS at 37°C in 5% CO 2 atmosphere. Buffy coats from healthy donors were obtained freshly from DRK-Blutspendedienst.
Orangu assay was purchased from Cell guidance systems. Human

| Isolation of human CD14 + cells
Human peripheral blood mononuclear cells (PBMC) were isolated from fresh buffy coats by density gradient. Therefore, 25

| Cell viability assay
For determination of the cell viability, Orangu assay was used according to the manufactory guidelines. Briefly, 1 × 10 5 monocytes were seeded in 96-well plates and incubated with various concentrations of silvestrol, with vehicle (DMSO) or were left untreated. After 30 minutes incubation (37°C, 5% CO 2 ), 10 ng/mL GM-CSF and 10 ng/mL IL-4 were added to differentiate MdDCs or 10 ng/mL GM-CSF to differentiate MdMs. For MdMs, the medium was completely refreshed after 3 days of incubation. After 48 hours (monocytes), 5 days (MdDCs) or 7 days (MdMs) 10 µL of Orangu™ cell counting solution was added to the wells and incubated for 120 minutes (37°C, 5% CO 2 ). Absorbance was measured at 450 nm with 650 nm as reference using EnSpire Plate Reader (PerkinElmer). Sample values were corrected with the background wells containing only medium without cells. Absorbance from treated cells was set in correlation to untreated cells. After differentiation, cells were centrifuged (300 g, 5 minutes, RT) and supernatant was stored for cytokine and chemokine detection at −80°C. Cells were washed with PBS, harvested with Accutase ® solution (15 minutes, 37°C, 5% CO 2 ) and cell count was determined using MACSQuant ® Analyzer 10. and cell count was determined using MACSQuant ® Analyzer 10.

| Activation of human dendritic cells
For differentiating monocytes to dendritic cells, 1.5 × 10 7 isolated CD14 + cells were seeded in T-75 flasks with 50 ng/mL of human GM-CSF and 50 ng/mL of human IL-4. After 5 days of differentiation without silvestrol, cells were harvested and seeded in triplicate, with 0.9 × 10 6 cells/well in 48-well plates. Silvestrol (0.5-5 nmol/L) or vehicle (DMSO) was added, and after 30 minutes of pre-incubation (37°C, 5% CO 2 ), 5 ng/mL human TNF-α, 5 ng/mL human IL-6, 5 ng/ mL human IL-1β, and 500 ng/mL PGE 2 were added. Cells were incubated for 24 hours (37°C, 5% CO 2 ) and harvested for analysis via flow cytometry. Supernatants were stored at −80°C for cytokine analysis. Fold induction of surface marker expression was calculated using the DMSO-treated cells as control. or ELISA for CCL18, CCL17 and IL-23 was performed. The cytometric bead array and the ELISA were performed according to the manufactory protocol.

| Determination of energy metabolism
For the analysis of the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) for human monocytes, macrophages and dendritic cells, the Seahorse XFe96 FluxPak (Agilent) was used as recommended by the manufacturer. CD14 + cells were isolated and human monocytes were cultivated for 48 hours without further differentiation factors while macrophages and dendritic cells were differentiated as described before. All cells were cultivated in the presence of 5 nmol/L silvestrol during the experiment. After differentiation, cells were washed with Seahorse XF RPMI medium pH 7.4 (Agilent), incubated for 60 minutes at 37°C, and OCR and ECAR were measured for a total period of 160 minutes in the absence of silvestrol. Cells were stimulated after 30 minutes. Monocytes were stimulated with 100 ng/mL lipopolysaccharides (LPS) and 20 ng/ mL IFN-γ, macrophages were stimulated with 20 ng/mL IFN-γ while dendritic cells were stimulated with a Stimulation-Mix containing 5 ng/mL of human TNF-α, IL-6, IL-1β and 500 ng/mL PGE 2 . Cells were measured as octuplicates (3 × 10 4 cells per well) using the Seahorse XFe96 Analyzer (Agilent) and analysed by Wave Software (Agilent).

| Statistics
Results are presented as means ± standard errors (SEM). For all calculations and creation of graphs, GraphPad Prism 8 was used and P < .05 was considered as the threshold for significance. Applied statistical analysis is denoted in the figure legends. In every test, silvestrol treatment was compared to vehicle.

| Silvestrol and cell viability
Since viable cells are a prerequisite for further experiments, the influence of silvestrol on cell viability was examined first. Using the Orangu assay, the percentage of viable cells after silvestrol or vehicle (DMSO) immune sensor for bacteria, was significantly increased at 5 nmol/L silvestrol, whereas the pattern recognition receptor CD206 and TREM2 were significantly reduced ( Figure 1D). Proinflammatory surface markers CD80, CD86 and HLA-DR were not significantly altered at the silvestrol concentrations used ( Figure S1A). Interestingly, silvestrol significantly reduced the release of the anti-inflammatory cytokine IL-10, the pro-inflammatory cytokine IL-6 and the chemokines CCL17 and CCL18 even at concentrations of 0.5 nmol/L ( Figure 1E). These data indicate that silvestrol alters the release of cytokines/chemokines and the expression of surface markers in differentiating macrophages, without generation of a recognized macrophage phenotype.

| Silvestrol reduces release of chemotaxins but promotes inflammatory markers in M1 MdMs
Next, we investigated whether silvestrol influences the polariza- and HLA-DR were not significantly changed at 5 nmol/L silvestrol ( Figure S2A). However, silvestrol significantly reduced the release of IL-10, and the chemotactic chemokines IL-8 and CCL2, whereas TNF-α was significantly increased in M1 MdMs ( Figure 2B). The release of CXCL10 and IL-23 was not influenced by silvestrol ( Figure 2B). These data indicate that silvestrol could potentially impair the recruitment of further immune cells as a result of the reduction of IL-8 and CCL2 release, whereas it might promote a pro-inflammatory environment by increasing TNF-α and decreasing IL-10 release.

| Silvestrol influences the differentiation of monocytes to MdDCs
Because not only macrophages but also dendritic cells are important players in the immune response to pathogens, the effects of silvestrol on dendritic cell differentiation were analysed. For this purpose, mono-  Figure 3A). The surface markers CD80 and CD197 were not modified ( Figure S4A).
Silvestrol led to a concentration-dependent reduction of IL-10, IL-8 and IL-6 release. IL-1β release was significantly increased after differentiation of MdDCs by silvestrol ( Figure 3B). IL-12 was not detectable either in vehicle or in silvestrol-treated MdDCs, and IL-23 release was not affected ( Figure S4B). These data indicate that silvestrol has a predominantly inhibitory effect on markers of the differentiation status of dendritic cells, but enhances IL-1ß release as well as CD141 and CD40 expression, at least partially reflecting potential promotion of cytotoxic T-cell responses, for instance to combat viral infections. 27,28

| Silvestrol influences the activation of dendritic cells
Next, we investigated whether silvestrol influences the activation of dendritic cells. Therefore, MdDCs were activated by IL-1ß, TNF-

| Silvestrol reduces energy metabolism
Because silvestrol had considerable impact on macrophages and dendritic cells, we investigated whether this might be a result of modified energy metabolism. Therefore, ECAR, a marker for glycolysis, and OCR, a marker for mitochondrial respiration, were de-

| D ISCUSS I ON
We  during dendritic cell differentiation, IL-6 and IL-10 release were both reduced by silvestrol. Additionally, it has been shown that silvestrol also inhibits phosphorylation of STAT-3, which would lead to opposite effects. 36 Therefore, another signalling pathway might be involved. In tumour models, silvestrol reduced STAT-1 mRNA. 17,36 Because STAT-1 is essential for DC differentiation, inhibition by silvestrol might explain the observed effects on reduced dendritic cell differentiation and activation. 41,42 Silvestrol may also modify immune cell infiltration by reducing release of chemokines and by down-regulation of adhesion molecules. CD54, a surface protein of the integrin family, was reduced by silvestrol during dendritic cell differentiation. This is in line with other antipathogenic drugs such as macrolides that also decrease the expression of CD54 43,44 and inhibit neutrophil migration. 45 Moreover, silvestrol reduced the release of chemotactic CCL18 and CCL17 during macrophage differentiation, of CXCL8 and CCL2 in M1 and M2 macrophages and of CXCL8 during dendritic cell differentiation and activation. Similarly, tetracyclines down-regulate the production of LPS-induced chemokines, such as CXCL8, CCL3 and CCL4 in THP-1 cells via NFκB signalling pathways. 46 The antiviral drug acyclovir inhibits the migratory potential of breast cancer cells. 47 These data indicate that silvestrol possibly attenuates inflammation by a reduction of immune cell attraction to the lesion site.
Our data further reveal that silvestrol impairs energy metabolism in myeloid cells. In this respect, it is noteworthy that silvestrol inhibits the proviral integration site for moloney murine leukaemia virus (PIM)1 and PIM2-two kinases involved among others in mechanisms of energy metabolism. 48 PIM inhibition via the mechanistic target of rapamycin complex 1 (mTORC1) pathway leads to reduced glycolysis in mouse embryonic fibroblasts. 49

CO N FLI C T O F I NTE R E S T
The authors declare no commercial or financial conflict of interest.