Immune modulatory effect of a novel 4,5-dihydroxy-3,3´,4´-trimethoxybibenzyl from Dendrobium lindleyi

Dendrobium bibenzyls and phenanthrenes such as chrysotoxine, cypripedin, gigantol and moscatilin have been reported to show promising inhibitory effects on lung cancer growth and metastasis in ex vivo human cell line models, suggesting their potential for clinical application in patients with lung cancer. However, it remains to be determined whether these therapeutic effects can be also seen in primary human cells and/or in vivo. In this study, we comparatively investigated the immune modulatory effects of bibenzyls and phenanthrenes, including a novel Dendrobium bibenzyl derivative, in primary human monocytes. All compounds were isolated and purified from a Thai orchid Dendrobium lindleyi Steud, a new source of therapeutic compounds with promising potential of tissue culture production. We detected increased frequencies of TNF- and IL-6-expressing monocytes after treatment with gigantol and cypripedin, whereas chrysotoxine and moscatilin did not alter the expression of these cytokines in monocytes. Interestingly, the new 4,5-dihydroxy-3,3′,4′-trimethoxybibenzyl derivative showed dose-dependent immune modulatory effects in lipopolysaccharide (LPS)-treated CD14lo and CD14hi monocytes. Together, our findings show immune modulatory effects of the new bibenzyl derivative from Dendrobium lindleyi on different monocyte sub-populations. However, therapeutic consequences of these different monocyte populations on human diseases including cancer remain to be investigated.


Introduction
The Dendrobium plants are vastly distributed in a large area including tropical and subtropical regions of Asia and North Australia [1]. Because of the extensive geographical distribution, Dendrobium becomes one of the largest genera in Orchidaceae, composing of approximately knowledge, chemical constituents and biological activities of compounds isolated from this orchid have never been investigated before. To assess their immune modulatory activities on circulating monocytes of the peripheral blood, we established an ex vivo model of primary human monocytes with low cell attachment, in which we attempted to minimize differentiation of monocyte into macrophage ex vivo. Our results revealed increased frequencies of TNF-and IL-6-expressing monocytes after treatment with gigantol and cypripedin, whereas chrysotoxine and moscatilin did not alter the expression of these cytokines in monocytes. Interestingly, the new 4,5-dihydroxy-3,3 0 ,4 0 -trimethoxybibenzyl derivative alleviated lipopolysaccharide (LPS)-induced cytokine production of primary human monocytes, suggesting immune modulatory activity.

Plant material
The

Buffy coats and monocyte isolation
Buffy coats from healthy individuals were obtained from the German Red Cross (GRC). Blood donors gave informed consent to perform blood collection and to use the buffy coats samples for research. The research purpose was approved by the Ethics Committee of Charité -Universitätsmedizin Berlin.
Human peripheral blood mononuclear cells (PBMCs) were isolated and aliquoted at 20 x 10 6 PBMCs (per mL) were cryopreserved in liquid nitrogen tank until analysis. Frozen PBMCs of three biologically independent donors were thawed, washed and pooled in MACS buffer (0.5% BSA in PBS containing 2 mM EDTA). Monocytes were isolated using negative selection, pan-monocyte Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer's specifications. Briefly, PBMCs were resuspended in 120 μL of MACS buffer. Thirty micro liters of FcR blocking reagent and 30 μL of biotin-antibody cocktail were added, mixed thoroughly and incubated at 4˚C. After 5 min incubation, 90 μL of MACS buffer and 60 μL of anti-biotin micro beads were added and incubated at 4˚C for 10 min. Stained ells were then washed with MACS buffer and pelleted (4˚C, 300 x g, 8 min). The pellet was then resuspended in 500 μL of MACS buffer and loaded onto the MACS column. The column was then washed twice with MACS buffer. The flow-through and washed fraction containing unlabeled monocytes was collected. Cell number and viability were determined by 0.2% trypan blue staining.

Cell culture and stimulation
Isolated monocytes were resuspended in 1 mL of RPMI1640 (Biochrom GmbH, Berlin, Germany) containing 10% heat-inactivated fetal calf serum (FCS) (Sigma-Aldrich, St. Louis, USA), penicillin (100 U/mL; Biochrom GmbH, Berlin, Germany) and streptomycin (100μg/ mL; Biochrom GmbH, Berlin, Germany). Cell concentration was adjusted to~2 x 10 6 cells/ mL. About 2 x 10 5 cells (per well) were transferred into ultra-low-attachment 96-well plate (Corning, New York, USA). Different concentrations of compounds ( Fig 3A) were added to the cell culture. To inhibit protein transport from Golgi apparatus to the endoplasmic reticulum, monensin (5 μg/mL; BioLegend, San Diego, USA) was also added right after the application of Dendrobium compounds. After two hours incubation, cells were stimulated by adding 100 ng/mL of lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, USA). Cells were cultured for 4 more hours or overnight (18 hours). Finally, cells were analysed by flow cytometry. Control conditions were naïve cells (without treatment of Dendrobium compounds and LPS) and unstimulated cells that were previously treated with the Dendrobium compounds.

Statistical analysis
Acquired FACS data were extracted in the Flow Cytometry Standard ( � .fcs) format. Cell populations were gated in FlowJo (Becton, Dickinson and Company, New Jersey, USA). Frequency of cells positive for a given signal was extracted into GraphPad Prism 8 (GraphPad Software Inc., California, USA) and statistically analyzed by a one-way ANOVA or a multiple t-test (as indicated), with a confidence interval of 95% (α = 0.05).

Establishment of low-attachment primary human monocyte cultures for functional analysis
It has been demonstrated that ex vivo culture of human monocytes using classical polypropylene cell culture plates resulted in phenotypic and functional changes, such as an increased granularity and reduced transendothelial diapedesis function [24]. Therefore, we newly established an ex vivo culture system using an ultra-low attachment multiple well-plate ( Fig 1A). First, we used peripheral blood mononuclear cells (PBMCs) to optimize the culture system ( Fig 1A and 1B). We observed a better preservation of the cell composition in the culture system using RPMI-1640 medium compared with reduced serum medium OptiMEM (Fig 1C  and 1D).
Next, we tested the LPS-induced inflammatory responses of monocytes under these culture conditions. We isolated monocytes (both CD14 lo and CD14 hi monocytes) from PBMCs using magnetic activated cell sorting (MACS) (Figs 1A and 2A). Isolated monocytes were then cultured in the established low-attachment culture system in the presence of 100 ng/mL LPS or PBS (control) for 6 hours. Compared to control, the cell composition of the monocyte population was changed after LPS stimulation. Namely, a strong reduction of CD14 + CD16 + monocytes and a moderately decreased frequency of CD14 hi monocytes were found ( Fig 2B). As expected, increased expression of TNF was detected in all three monocyte populations after LPS stimulation, with the highest response in CD14 hi inflammatory monocytes (Fig 2C and  2D). Furthermore, we found increased expression of IL-6 in CD14 hi and CD14 lo monocytes after LPS stimulation, whereas MCP-1 (or CCL2) and CD68 were unchanged under these conditions (Fig 2E and 2F).

Evaluation of immune modulatory effects of Dendrobium compounds
To assess immune modulatory effects of all five Dendrobium compounds, we cultured MACSisolated primary human monocytes under our established culture conditions (Figs 1 and 2), including an inhibitor of intracellular protein transport-monensin, in the presence of Dendrobium compounds, which were 4,5-dihydroxy-3,3 0 ,4 0 -trimethoxybibenzyl (#1), chrysotoxine (#2), gigantol (#3), cypripedin (#4) and moscatilin (#5). Since these five compounds were diluted in dimethyl sulfoxide (DMSO), the same concentration of DMSO was added to the control culture without any compounds. We tested three known therapeutic concentrations [5][6][7][8][9], which were 5, 10 and 20 μM. After 2 hours of incubation, we stimulated monocytes by adding 100 ng/mL LPS and incubated for four more hours. We observed an increased frequency of TNF-expressing cells in gigantol (#3)-and cypripedin (#4)-treated monocytes without LPS stimulation, suggesting immune modulatory effects of these compounds on monocyte responses ex vivo (Fig 4A and 4B). After LPS stimulation, we detected increased frequencies of TNF-and IL-6-expressing monocytes, which were most pronounced in CD14 hi inflammatory monocytes (Fig 4A and 4B), which is in line with the results shown in Fig 2. Although we observed decreased frequencies of LPS-induced TNF-and IL-6-expressing cells in monocytes treated with 4,5-dihydroxy-3,3 0 ,4 0 -trimethoxybibenzyl (#1), chrysotoxine (#2) and moscatilin (#5), only the compound #1 showed promising dose-dependent positive effects (Fig 4A-4D). Therefore, we selected the compound #1 for a further evaluation. We tested whether the immune modulatory effects of Dendrobium compounds on monocytes persisted also after long-term exposure to LPS. To do so, the isolated monocytes were first incubated in the presence of the compound #1 for two hours followed by overnight LPS-stimulation in a presence of monensin (5 μg/mL). We observed decreased expression of CD14 of the overnight cultured monocytes, and an increased abundance of TNF-and IL-6-expressing CD11c lo monocytes (Fig 5A), especially in the CD14 hi HLA-DR + sub-population. Overnight exposure of monocytes to compound #1 resulted in changes in the monocyte composition, indicating as an increased abundance of CD14 lo HLA-DRand CD14 lo HLA-DR + cells and a decreased abundance of CD14 hi HLA-DR + cells (Fig 5B). These changes were found at a highest degree in monocytes treated with 20 μM of #1. Furthermore, these observed changes were more pronounced in LPS-stimulated monocytes (Fig 5B, +LPS). Similar to the short-term incubation (Fig 4), CD14 hi monocytes were the main source of TNF-and IL-6-expressing cells (Fig 5A, 5C & 5D). Overnight incubation with 20 μM of the compound #1 alone induced TNF expression in CD14 lo HLA-DR + and CD14 lo HLA-DRcells (Fig 5C), while under LPS-stimulation it was downmodulating the LPS-induced TNF-expression (Fig 5C). In comparison to the control cells (without the compound #1), a treatment with 10 μM of #1 could decreased LPS-induced TNF expression in all monocyte subsets (Fig 5C). The treatments with either 5 or 20 μM of #1 could only decreased the LPS-induced TNF expression in CD14 hi HLA-DR + monocytes ( Fig  5C). Without LPS-stimulation, the compound #1 induced IL-6 expression in both CD14 lo and CD14 hi HLA-DR + monocytes, but not in CD14 lo HLA-DRcells ( Fig 5D). Interestingly, the 5 and 10 μM of the compound #1 strongly induced IL-6 expression in all monocyte subsets under LPS stimulation conditions, whereas the 20 μM concentration did not affect the expression of IL-6 in all three subsets (Fig 5D). The findings demonstrate a dose-dependent activating and/or immune modulatory effects of the compound #1, which are observed to be different in different monocyte sub-populations.

Discussion
In this study, we demonstrated the use of low-attached culture model of primary human monocytes as a tool to study immune modulatory effects of purified natural products (the Dendrobium compounds). We showed the immune modulatory potential of a novel Dendrobium compound: 4,5-dihydroxy-3,3 0 ,4 0 -trimethoxybibenzyl, which appeared to be dose-dependent and different between monocyte subsets. In addition, we have tested those known compounds including chrysotoxine (#2), gigantol (#3), cypripedin (#4) and moscatilin (#5), which were proposed as potential candidates for the development of cancer therapy. However, these compounds appeared to have either less immune modulatory activity or even more inflammatory effect on primary monocytes, compared with the compound #1. Our findings suggest an importance and/or feasibility of using ex vivo studied models of different primary human (immune) cells to estimate the therapeutic potential and/or any potential adverse effects of purified natural products, which will further facilitate the adjustment of developmental potential of each isolated compound in diseases.
In the low-attached monocyte culture model used in this study, we found stronger responses to TLR4 agonist LPS of CD14 hi monocytes, compared to CD14 lo monocytes, on the basis of the expression of inflammatory cytokines especially TNF and IL-6. Our results are in line with the study by Cros et al. in which CD14 dim was demonstrated to respond poorly to agonists of TLR1, TLR2 and TLR4 [34]. The inflammatory cytokine TNF has been shown to be involved in many biological processes including fever, apoptosis, and infection-induced cachexia [16], as well as in inflammation-associated diseases, for example rheumatoid arthritis [35], acquired generalized lipodystrophy and combined Crohn's disease [36], Crohn's disease [37], and type 2 diabetes [38]. Moreover, chemokine such as TNF can also induce activate inflammatory responses, and are implicated in the regulation of tumor development and growth via regulation of tumor-associated angiogenesis, by activation of host immunological Multiple t-test, corrected for multiple comparisons using Holm-Sidak method, � P < 0.05, �� P < 0.01, ��� P < 0.001, ���� P < 0.0001. (C) Histograms demonstrate fluorescent intensity representing LPS-induced TNF expression in different monocyte subsets, compared to naïve monocytes (unstimulated, PBS-treated). (D) Bar graphs show quantitative analysis of fluorescent intensity representing LPS-induced TNF expression in different monocyte subsets, compared to naïve monocytes (unstimulated, PBS-treated). Each dot represents an independent measurement. One-way ANOVA, corrected for multiple comparisons using Tukey Test, � P < 0.05, �� P < 0.01, ��� P < 0.001, ���� P < 0.0001. (E) Histograms demonstrate fluorescent intensity representing LPS-induced cytokine expression of CD14 hi monocytes (red line), compared to naïve CD14 hi monocytes (unstimulated, PBS-treated; dashed line). (F) Bar graphs show quantitative analysis of fluorescent intensity representing LPS-induced cytokine expression in different monocyte subsets, compared to naïve monocytes (unstimulated, PBS-treated). One-way ANOVA, corrected for multiple comparisons using Tukey Test, � P < 0.05, �� P < 0.01, ��� P < 0.001, ���� P < 0.0001.
https://doi.org/10.1371/journal.pone.0238509.g002 responses or by direct inhibition of tumor cell proliferation [39]. It has been also shown that suppressing the increased expression of inflammation-related factors such as TNF is considered to potentially suppress the migration of macrophages into tumor tissues and regulate inflammatory changes in the tumor environments, respectively [40]. In this study, we detected Dendrobium compound-induced TNF expression in monocytes treated with 20 μM of gigantol (#3) or cypripedin (#4), suggesting potentially inflammatory effects of these compounds at high concentration. Whereas 4,5-dihydroxy-3,3 0 ,4 0 -trimethoxybibenzyl (#1), chrysotoxine (#2) and moscatilin (#5) did not show obvious inflammatory effects on monocytes in vitro, only the novel compound #1 has shown promising immune modulatory effects in a dosedependent manner. However, in a long-term LPS-stimulation, only the 10 μM of the compound #1 could downmodulate the LPS-induced TNF expression in all monocyte subsets, whereas the concentration of 20 μM induced TNF expression of unstimulated cells, suggesting dose-and treatment-duration-dependent immune modulatory effects and/or inflammatory effects of this compound. Interleukin-6 (IL-6) is a cytokine with pleiotropic function, which are involved in host defense. In acute infection and/or tissue injury, monocytes/macrophages promptly produce IL-6 and contribute to removal of infectious agents and regeneration of damaged tissue through activation of immune, hematological, and acute-phase responses [41]. However, a dysregulation of IL-6 production and/or persistently increased IL-6 expression plays a pathological role in the development of various inflammatory diseases and cancers, thus IL-6 is a double-edged sword for the host [41]. Thus, the proper IL-6 expression is very important for host defense. In this study, although we did observe the reduction of the LPS-induced IL-6 production in monocytes, that were short-term treated with the compound #1, we found strongly increased production of IL-6 in long-term treated monocytes (Fig 5). However, since we detected a decrease in LPS-induced TNF-expression under the same conditions, an increased IL-6 expression observed in this study may refer to increased monocyte activity against LPS-induced inflammation.

Conclusions
In summary, we demonstrate herein immune modulatory effects of the new compound 4,5-dihydroxy-3,3 0 ,4 0 -trimethoxybibenzyl on different monocyte subsets ex vivo, which can potentially be developed as in vivo immune modulatory drug in a broad spectrum of inflammation-driven diseases besides lung carcinomas. However, since Dendrobium lindleyi is widely distributed, using this orchid for drug development may have a risk of habitat destruction, over-collection and commercially trade due to low natural regeneration rates. Moreover, Dendrobium plants are listed on Appendix II (D. cruentum is the only species listed on Appendix I) of Convention on International Trade in Endangered Species of Wild Fauna and Flora [42], which monitors, regulates or bans the trade to ensure species survival [43]. Therefore, in parallel to drug development, an establishment and improvement of methodologies/techniques for seedling propagation and artificial cultivation technology such as tissue culture and artificialsheltered cultivation [44][45][46][47] are required to ensure sustainable development and future marketing. mean frequency (%) of CD14 lo and CD14 hi monocytes, which express TNF and IL-6, compared to naïve monocytes (no LPS). This is an explorative screening experiment, in which each condition (or each concentration) was analyzed in duplicate, thus no variation was shown. (C-D) The X-Y graphs show the correlation between the frequency (%) of TNF-or IL-6-expressing CD14 hi or CD14 lo monocytes and the concentration of all five Dendrobium compounds (#1 -#5). https://doi.org/10.1371/journal.pone.0238509.g004