The Multifaceted Effects of Polysaccharides Isolated from Dendrobium huoshanense on Immune Functions with the Induction of Interleukin-1 Receptor Antagonist (IL-1ra) in Monocytes

Dendrobium huoshanense is a valuable and versatile Chinese herbal medicine with the anecdotal claims of cancer prevention and anti-inflammation. However, its immunological activities are limited to in vitro studies on a few cytokines and immune cell functions. First, we investigated the effects of polysaccharides isolated from DH (DH-PS) on inducing a panel of cytokines/chemokines in mice in vivo and human in vitro. We found that DH polysaccharides (DH-PS) induced TH1, TH2, inflammatory cytokines and chemokines in mouse in vivo and human cells in vitro. Secondly, we demonstrated that DH-PS expanded mouse splenocytes in vivo including CD4+ T cells, CD8+ T cells, B cells, NK cells, NKT cells, monocytes/macrophages, granulocytes and regulatory T cells. Notably, DH-PS induced an anti-inflammatory molecule, IL-1ra, in mouse and human immune cells, especially monocytes. The serum level of IL-1ra elicited by the injection of DH-PS was over 10 folds of IL-1β, suggesting that DH-PS-induced anti-inflammatory activities might over-ride the inflammatory ones mediated by IL-1β. The signaling pathways of DH-PS-induced IL-1ra production was shown to involve ERK/ELK, p38 MAPK, PI3K and NFκB. Finally, we observed that IL-1ra level induced by DH-PS was significantly higher than that by F3, a polysaccharide extract isolated from another popular Chinese herbal medicine, Ganoderma lucidum. These results indicated that DH-PS might have potential applications for ameliorating IL-1-induced pathogenic conditions.


Introduction
Dendrobium huoshanense (DH), which is an herb of Orchidaceae family, has been used as a traditional Chinese herbal medicine for centuries with the anecdotal claims of cancer prevention and antiinflammation. Polysaccharides isolated from Dendrobium huoshanense have been reported to induce TNF-a in peritoneal macrophages and IFN-c in mouse splenocytes [1] and promote phagocytosis of macrophages [2]. To date, there have been no detailed studies on the systemic immune functions of DH-PS such as in vivo immune cell activations, inductions of comprehensive panel of cytokines/ chemokines and anti-inflammatory molecules.
Among the cytokines, two forms of Interleukin-1 (IL-1a and IL-1b) are thought to play an important role in inflammation and involved in many pathological conditions including rheumatoid arthritis [3,4]. They are produced primarily by mononuclear phagocytes, but also by a number of other cell types including skin keratinocytes [5]. These two cytokines are pro-inflammatory cytokines which can stimulate the expressions of genes associated with inflammation and autoimmune diseases. IL-1 exerts its functions by binding to type I IL-1 receptor and induces downstream signaling, leading to the expressions of numerous genes resulting in inflammation [6,7,8,9]. A natural inhibitor of IL-1 activity, designated as secreted Interleukin-1 receptor antagonist (IL-1ra), was discovered and purified from the urine of the patients suffering from monocytic leukemia [7,10]. IL-1ra, a 25 KD glycoprotein, is a member of IL-1 family that competes with IL-1 for the binding to IL-1 receptor, but unlike IL-1, this binding does not induce any signal transduction [11,12,13,14]. IL-1ra is released in vivo during inflammation and immune-mediated diseases [15], which is thought to limit the deleterious effects brought by IL-1 [16,17] and shown to be effective in the treatment of sepsis, graft-versus-host disease and rheumatoid arthritis in animal models [18,19,20,21]. Additionally, IL-1ra (commercially produced as ''anakinra'') has been used clinically to treat rheumatoid arthritis in which IL-1 plays a key role [22]. Many types of immune cells are reported to secrete IL-1ra including neutrophils, master cells, macrophages and monocytes [23,24,25] and several molecules have been shown to stimulate the secretion of IL-1ra including cytokines (IL-6 and IL-10, for example) and natural products [13,26]. Polysaccharides isolated from Ganoderma Lucidum, another popular Chinese herbal medicine, have been shown to possess immune-modulating effects and reported to induce the production of IL-1ra [27]. Although DH-PS is believed to possess immune-modulating functions with the anecdotal claims of cancer prevention and anti-inflammation, comprehensive information regarding DH-PS on immune functions and its ability to stimulate the secretion of IL-1ra is still lacking.
In the present study, we evaluated the immune-modulating effects of DH-PS by investigating the profiles and kinetics of cytokine productions, as well as expansions and activations of immune cells induced by DH-PS in both human and mouse system. Moreover, we demonstrated that DH-PS induced IL-1ra production through the activation of PI3K, p38 MAPK and NFkB. Finally, we compared the IL-1ra production induced by DH-PS and F3, the polysaccharide extract from another popular Chinese herbal medicine, Ganoderma lucidum.

DH-PS induced the secretions of multiple cytokines and chemokines in vivo
In order to understand the immunological activities of DH-PS, we investigated the profiles of cytokines and chemokines induced by DH-PS in mice. BALB/c mice (n = 3 for DH-PS and phosphate buffered saline (PBS) group) were injected intraperitoneally (IP) with DH-PS (300 mg/mouse) in PBS or PBS only and the sera were collected at 0 (before injection), 2 and 18 hrs after the injection of DH-PS for the detection of cytokines and chemokines, which were quantified by Beadlyte Mouse 21-plex Cytokine Detection system and ELISA kits (for IL-1ra). Compared with PBS group shown in Figure 1, the increase of TNF-a, IL-12 p40, IL-6, IL-10 and RANTES were observed at 2 hrs, along with robust inductions of KC, MCP-1 and MIP-1b. An apparent increase in IL-1b at 2 hrs was noted but it did not reach statistical significance. These results suggested that DH-PS induced Th1 (IL-12 p40), Th2 (IL-6, IL-10), inflammatory cytokines (TNF-a) and chemokines (KC, RANTES, MCP-1, MIP-1b), which might further modulate the downstream activations of immune cells. Furthermore, an antiinflammatory molecule, IL-1ra, was increased from 465 to 4199 pg/ml (,9 folds of PBS control group) at 2 hrs. This indicated that the amounts of IL-1ra induced were 10 folds more than IL-1b (increased from 144 to 397 pg/ml, ,1.6 folds of PBS control group), suggesting that DH-PS-induced IL-1ra might override the activity induced by IL-1b.

DH-PS expanded and activated subpopulations of immune cells in vivo
In order to investigate the effects of DH-PS on immune cells, we further analyzed whether DH-PS expanded and/or activated the subpopulations of mouse splenocytes. BALB/c mice (n = 3 for DH-PS and PBS group) were injected intraperitoneally with DH-PS (100 mg or 300 mg) or PBS (DH-PS = 0) only and sacrificed at 72 hrs for the harvest of splenocytes. Cells were counted and analyzed for markers characteristic of specific subpopulations of splenocytes by flow cytometry to determine the expansions/ activations of various immune effector cells. As shown in Figure 2A, in comparison with PBS control group, the number of splenocytes was increased after the administration of DH-PS in a dose-dependent manner. Subpopulations of splenocytes exam-ined included innate immune cells such as natural killer cells (NK-1.1 + /CD3 2 )/activated natural killer cells (NK-1.1 + /CD3 2 / CD69 + ), natural killer T cells (NK-1.1 + /CD3 + )/activated natural killer T cells (NK-1.1 + /CD3 + /CD69 + ), regulatory T cells (CD4 + / CD25 + /Foxp3 + ), granulocytes (Ly6G + ), monocytes and macrophages (CD11b + ) and dendritic cells (CD11c + /CD80 + /CD86 + ) and adaptive immune cells such as B cells (B220 + CD23 + )/ activated B cells (B220 + CD23 + CD69 + ), CD4 + T cells (CD3 + CD4+)/activated CD4 + T cells (CD3 + CD4 + CD69 + ), CD8 + T cells (CD3 + CD8 + )/activated CD8 + T cells (CD3 + CD8 + CD69 + ). Overall, DH-PS induced modest increases (up to 2.08 fold) in all subpopulations examined. Compared with PBS group, we found that DH-PS induced 1.3 and 1.7 fold increases of NK cells at 100 mg and 300 mg, respectively. A mild increase (1.24 folds) of activated NK cells was also observed at 300 mg (Fig.2B). DH-PS induced 1.3 and 1.6 fold increases of NKT cells at 100 mg and 300 mg, respectively. An increase (1.37 folds) of activated NKT cells was also observed at 300 mg (Fig.2C). DH-PS also induced 1.55 and 1.79 fold rise of regulatory T cells at 100 mg and 300 mg, respectively (Fig.2D) and a trend in increases of granulocytes and monocytes/macrophages (Fig.2E, F). However, there was no significant effect of DH-PS on dendritic cells (Fig.2G). For the adaptive immune cells, DH-PS induced 1.27 and 1.53 fold increases of B cells and 2.08 and 1.91 fold rise of activated B cells at 100 mg and 300 mg, respectively (Fig.2H). As to CD4 + T cells (Fig.2I), DH-PS induced 1.32 and 1.56 fold increases at 100 mg and 300 mg, respectively. Activated CD4 + T cells were also augmented to 1.64 and 1.98 folds, respectively. For CD8 + T cells (Fig.2J), DH-PS induced 1.22 and 1.50 fold increases and activated CD8 + T cells to 1.63 and 1.96 folds at 100 mg and 300 mg, respectively. These findings indicated that DH-PS modestly expanded and/or activated many types of immune cells in mice.

DH-PS induced the productions of multiple cytokines and chemokines in human immune cells
We next investigated the effects of DH-PS on primary human immune cells. Human peripheral blood mononuclear cells (PBMC) were isolated from three healthy donors and cultured with DH-PS (50 mg/ml) or PBS as control for 18 hrs. Supernatants were collected for the measurements of cytokines, chemokines and IL-1ra. As shown in Figure 3, TH1 (IL-12 p40), TH2 (IL-6, IL-10), inflammatory cytokines (IL-1a, IL-1b TNF-a) and chemokines (MIP-1a) were induced in PBMC by DH-PS. A growth factor GM-CSF was also augmented by DH-PS. Consistent with mouse data, IL-1ra was induced by DH-PS in human PBMC. Since PBMC contained a mixture of immune cells, we next focused on the DH-PS-induced effects on CD14 + cells which were reported to play a key role in rheumatoid arthritis. CD14 + cells were isolated from PBMC and cultured with DH-PS (50 mg/ml) or PBS as control for 18 hrs. The supernatants were collected for the measurements of cytokines, chemokines and IL-1ra. As shown in Figure 4, there were significant increases in IL-1a, IL-1b and IL-1ra along with IL-12 p40, IL-6, TNF-a, IL-10, GM-CSF, RANTES, MCP-1 and MIP-1a. Moreover, DH-PS boosted IL-1ra production from basal levels of (135-196) to 1427, 837 and 1264 (pg/ml) and induced IL-1b from 7 to 55, 24 and 70 (pg/ml) in CD14 + cells of 3 healthy donors. Thus the calculated IL-1ra/ IL-1b ratios were 25.9, 34.8 and 18.1, suggesting that the IL-1ra induced by DH-PS might over-ride IL-1b activities. These data indicated that DH-PS induced Th1, Th2, inflammatory cytokines, chemokines and an anti-inflammatory molecule, IL-1ra, with an overall anti-IL-1b activity and the pattern of cytokines and chemokines induced by DH-PS was similar to the results in mice.

Dose-dependency of IL-1ra induction by DH-PS in mice
As a potent antagonist of the biological functions of IL-1, IL-1ra has been in clinical use for the treatment of IL-1-induced pathogenic conditions including rheumatoid arthritis. To further delineate the kinetics and dose effects of DH-PS on IL-1ra production, BALB/c mice (n = 3 for each group) were injected intraperitoneally with DH-PS (100 or 300 mg/mouse) or PBS. Sera were collected at 0 (before injection), 2 and 18 hours for the measurements of IL-1ra. As shown in Figure 5, IL-1ra was significantly increased in a dose-dependent manner at 2 hrs after the administration of DH-PS, as compared to PBS group, and declined rapidly to basal level at 18 hrs.

DH-PS induced IL-1ra secretion in human monocytes but not neutrophils
Next, we investigated the effects of DH-PS on IL-1ra secretion in human primary immune cells. At the concentrations ranging from 0 (use of PBS as control) to 200 mg/ml, DH-PS induced dosedependent increases of IL-1ra production in PBMC, reaching the maximal level of ,10 fold rise at 100 mg/ml (Fig.6A). Since monocytes and neutrophils were two types of immune cells reported to produce IL-1ra upon stimulations [24], we further investigated the responses of the two types of cells stimulated by DH-PS. CD14 + cells were isolated from PBMC and cultured with increasing concentrations of DH-PS ranging from 0 (use of PBS as control) to 100 mg/ml for 18 hrs. As shown in Figure 6B, DH-PS induced CD14 + cells to produce IL-1ra dose-dependently, reaching the maximal level of ,6 fold increases at 100 mg/ml. However, neutrophils showed no significant production of IL-1ra with the stimulation of DH-PS for 3 or 24 hrs (Fig.6C). Taken together, DH-PS stimulated IL-1ra production in human CD14 + cells but not neutrophils.
Next, we used a human monocytic cell line, THP-1, as our model for more detailed studies. THP-1 cells were seeded at different densities and cultured with increasing concentrations of DH-PS for 18 hr. As shown in Figure 7A, the production of IL-1ra was dose-dependent starting from 1 mg/ml. The kinetics of IL-1ra induced by DH-PS was examined in THP-1 cells cultured at 2610 6 cells/ml with DH-PS (100 mg/ml) or PBS as control. The supernatants were collected for IL-1ra measurement at various time points. As shown in Figure 7B, DH-PS induced 2.2 fold increases of IL-1ra production at 3 hrs and the induction steadily increased to 72 hrs. The kinetics of IL-1ra induced by DH-PS at the RNA level was also examined by RT-PCR. As shown in Figure 7C, IL-1ra mRNA became detectable at 1 hr and appeared to reach the plateau at 12 hrs. In addition to the induction of IL-1ra in THP-1 cells, there was a dose-dependent increase in cell

Intracellular signaling of DH-PS-induced IL-1ra production in monocytes
To investigate DH-PS-induced intracellular signaling mediating IL-1ra expression, we utilized inhibitors for various kinases including ERK/ELK (PD98059), JNK (SP600125), p38 MAPK (SB203580), PI3K (Ly294002) and NFkB inhibitors (Helenalin and MG132) at concentrations showing no toxicity to cells (Fig.S1). THP-1 cells (Fig.8A) or human CD14 + cells (Fig.8B) were pretreated with these inhibitors at increasing concentrations or DMSO as control for 60 minutes and cultured with DH-PS (100 mg/ml) for another 18 hrs. In both THP-1 and human CD14 + cells, the inhibitors of ERK/ELK, JNK, p38 MAPK, PI3K and NFkB diminished the secretion of IL-1ra in a dosedependent manner. In order to determine whether these signaling molecules regulated the IL-1ra production at transcriptional level, THP-1 cells were pretreated with the inhibitors at indicated concentrations or DMSO as control for 60 minutes and cultured with DH-PS (100 mg/ml) for another 12 hrs. The mRNA expression level of IL-1ra was examined by RT-PCR. The results showed that inhibitors of ERK/ELK, PI3K and NFkB (Helenalin) dampened IL-1ra mRNA expression (Fig.8C), which was consis-tent with the results of IL-1ra production in protein level (Fig.8A). On the other hand, the inhibitors of JNK and p38 MAPK had no significant effects on mRNA expression.

DH-PS induced larger amounts of IL-1ra than F3 (G. lucidum)
Ganoderma lucidum is a Chinese herbal medicine that has been used for centries to treat a variety of diseases including inflammation and cancer [28]. F3, the polysaccharide extract of Ganoderma lucidum has been reported to possess immune-modulating functions and induced IL-1ra in mice [27]. Therefore, we examined the induction of IL-1ra by DH-PS or F3 in human CD14 + cells and THP-1 cells and the kinetics of IL-1ra mRNA expression in THP-1 cells. Human CD14 + cells (Fig.9A) and THP-1 cells (Fig.9C) were cultured with increasing concentrations of DH-PS or F3 for 18 hrs. The kinetics of IL-1ra secretion in CD14 + cells upon the treatments of DH-PS and F3 (100 mg/ml) was also measured (Fig.9B). As shown in Figure 9A and 9C, both DH-PS and F3 induced dose-dependent productions of IL-1ra, but the maximal level induced by DH-PS was 2.2 folds of that by F3 in both CD14 + and THP-1 cells. As to the kinetics of IL-1ra induction in CD14 + cells, DH-PS elicited faster and larger amounts of IL-1ra than F3 with 1.4, 1.7 and 2.0 folds at 3, 24 and 48 hrs, respectively, reaching 2.1 folds at 72 hrs (Fig.9B). We also assessed the kinetics of IL-1ra mRNA expression in THP-1 cells cultured with DH-PS or F3. As shown in Figure 9D, DH-PS induced higher IL-1ra mRNA expression than F3, which was consistent with the ELISA data. On the other hand, F3 induced larger amounts of IL-1b (366 pg/ml, ,52 fold increases of PBS control) (Fig.S3) than DH-PS in human CD14 + cells (55, 24, 70 pg/ml for 3 healthy donors respectively, Fig.4). Taken together, DH-PS-induced IL-1ra rise could over-ride IL1binduced activity far more effectively than F3.

Discussion
Dendrobium huoshanense (DH) is a versatile and valuable Chinese herbal medicine that has been used for a long period of time in China. The main bioactive molecules are polysaccharides and alkaloids. Since it is highly valuable but rare, strategies have been developed to enhance its growth and increases in polysaccharides production [29]. Using crude extracts of polysaccharides isolated from DH, we previously showed the in vitro induction of G-CSF and a few other cytokines in mouse splenocytes [30]. Our present study further extended the investigation to cover a comprehensive panel of cytokines and chemokines induced by DH-PS in immune cells of human in vitro and mouse in vivo. Our results clearly showed that the administration of DH-PS in mice modulated immune functions through modest activation and/or expansion of various immune cells including NK cells/activated NK cells, NKT cells/ activated NKT cells, regulatory T cells, B cells/activated B cells, CD4 + T cells/activated CD4 + T cells and CD8 + T cells/activated CD8 + T cells. This was accompanied by the production of Th1 (IL-12 p40), Th2 cytokines (IL-6 and IL-10), chemokines (KC, MCP-1, MIP-1b, RANTES) and inflammatory cytokines (TNF-a). We also provided the first evidence that many cytokines and chemokines were induced by DH-PS in human PBMC (IL-1a, IL-1b, IL-12 p40, IL-6, IL-10, TNF-a, MIP-1a GM-CSF and IL-1ra) and monocytes (IL-1a, IL-1b, IL-12 p40, IL-6, IL-10, TNF-a, RANTES, MCP-1, MIP-1a, GM-CSF and IL-1ra). Notably, both IL-6 and IL-10 which have been shown to stimulate the production of IL-1ra were also induced by DH-PS. These findings were reminiscent of the potent immune-modulating effects induced by Ganoderma lucidum (Reishi), another popular Chinese herbal medicine. It was reported that polysaccharides isolated from Ganoderma lucidum (Reishi) enhanced the proliferation of Con A-stimulated mouse splenocytes [31]. We also showed that F3 stimulated the expansions of several types of immune cells including regulatory T cells [32]. But unlike DH-PS, F3 appeared to induce fewer regulatory T cells than DH-PS and larger amounts of IFN-r and IL-12 p70 which were below the detection limit upon the treatment with DH-PS, suggesting that DH-PS might exert greater anti-inflammatory activities. Taken together, DH-PS might exert its immune modulations not only by directly stimulating cytokine secretions but also through promoting the expansions and/or activations of immune cells.
Another important anti-inflammatory molecule found to be induced by DH-PS was IL-1ra which was an acute phase protein [33] and often elevated in the peripheral blood of patients with sepsis [34], chronic rheumatic diseases [35,36,37] and following surgical trauma [38,39]. The beneficial effects of IL-1ra on inflammatory disorders had been demonstrated in many experimental animal models of disease by administration of recombinant IL-1ra [26,40,41]. In fact, recombinant IL-1ra had been in clinical use for sepsis syndrome [42,43] and rheumatoid arthritis. Many molecules including cytokines (IL-6 and IL-10, for example) and bglucans [26] which were the backbone components of the main  bioactive polysaccharides of G. lucidum were reported to induce IL-1ra production. Here, we demonstrated for the first time that DH-PS induced the production of IL-1ra both in vivo and in vitro. The rapid induction of IL-1ra by DH-PS in both mouse and human suggested a direct stimulation by DH-PS. Many types of immune cells were known to secrete IL-1ra including macrophages, mast cells, neutrophils and monocytes, and our results showed that DH-PS stimulated the production of IL-1ra in monocytes but not neutrophils. We also found that unlike in vitro cell experiments, the level of IL-1ra in sera declined rapidly at 18 hrs after the treatment of DH-PS. This is consistent with the known short biological halflife of IL-1ra in vivo with rapid renal clearance and excretion in the urine [44]. In addition, DH-PS promoted THP-1 cell expansions dose-dependently, suggesting that DH-PS might also stimulate the proliferation of cells. Since certain glycans were the ligands for Toll-Like Receptors (TLRs) which were important for the activation of monocytes and involved in the transcription of IL-1ra [25,45], it will be interesting to delineate whether and which TLRs are the receptors for DH-PS in the future.
There were several intracellular signaling mechanism reported to be involved in the regulation of IL-1ra expression. For examples, NF-kB and C/EBP had been shown to be involved in the expression of IL-1ra gene in hepatocytes [33], and MAPK (ERK1/ERK2) was associated with the IL-1ra induction stimulated by LPS [46,47]. Serine/threonine phosphatases also participated in IL-1ra production when monocytes were contacted by stimulated T cells [48]. In addition, STAT6 was involved in IL-4 mediated IL-1ra production [49] and PI3K was reported to be essential for the IFN-b-mediated IL-1ra production [50]. Our results suggested that ERK/ELK, p38 MAPK, PI3K and NF-kB were involved in DH-PS mediated production of IL-1ra since their specific inhibitors decreased the expression of IL-1ra mRNA and the IL-1ra production in protein level. Our results suggested several possible signaling pathways involved in DH-PS-induced IL-1ra secretion.
Ganoderma lucidum (Reishi) has been known for its benefits in human health with the possession of anti-tumor and immunemodulating activities [51,52,53,54]. The polysaccharides isolated from Reishi were composed of a branched (1-.6)-b-D-glucan moiety. The main structure of polysaccharides isolated from DH-PS had been confirmed as acetylated glucomannan [30] which is different from Reishi. The effects of Reishi on the immune system had been attributed to cytokine inductions [55]. We compared the cytokine profiles of human monocytes cultured with DH-PS or F3 and found that both DH-PS and F3 induced several cytokines and chemokines including IL-1b, TNF-a, GM-CSF, IL-12 p40, RANTES, MCP-1, MIP-1a, IL-6 and IL-10. However, IL-12 p70 and IFN-c were induced only by F3, suggesting a more TH1 bias activity of F3 compared with DH-PS since IL-12 p70 and IFN-c had been reported to contribute to the differentiation of type 1 T helper cell [56]. The ability of F3 to induce the production of IL-1ra has also been reported [27]. However, we found that DH-PS induced higher IL-1ra and lower IL-1b than F3 in human CD14 + cells and THP-1 cells Collectively, our findings of lower levels of IL-12 p70, IFN-c, IL-1b, larger amounts of IL-1ra and more regulatory T cells induced by DH-PS suggested that DH-PS might possess better anti-inflammatory activities than F3. Thus, DH-PS might be more potent than F3 for alleviation of inflammatory disorders. Since the main structure of DH-PS had been reported as acetylated glucomannan, it will be worthwhile to further identify the specific glycan moieties responsible for its antiinflammatory effects and its therapeutic potential in certain immune disease models.

Ethics statement
Normal human blood was obtained from Taipei Blood Center with the approval of the Human Subject Research Ethics committee of both Academia Sinica and the Taiwan Blood Services Foundation. All participants provided written informed consent to Taipei Blood Services Foundation. All animal studies were performed under the approved protocol #TMIZ00JY2005158 by Institutional Animal Care and Utilization Committee of Academia Sinica.

Preparations for crude polysaccharide extracts from Dendrobium huoshanense (DH-PS)
The plant material of D. huoshanense was obtained from Yuen-Foong-Yu Biotech Co. Taiwan [30]. Non-lignified primary mucilage polysaccharides were collected from ground leaves and stems at 4uC by dd-H 2 O extraction and the extracts were filtrated to remove the insoluble parts. The dd-H 2 O extractions (DH-PS) were dried for storage and resuspended in PBS before animal or cell experiments.

Cell culture and reagents
Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors by Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) according to manufacturer's suggestions. CD14 + cells were further purified from PBMC by anti-CD14 microbeads and magnetically activated cell sorting (MACS) system (Miltenyi Biotech, Auburn, CA) according to manufacturer's instruction. PBMC and purified CD14 + cells (purity .90%) were cultured in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 10% heat-inactivated Fetal bovine serum (FBS) (Sigma-Aldrich, USA) and penicillin/streptomycin (100 units/ml) (Invitrogen, CA, USA). For isolation of neutrophils from healthy donors, leukocytes (including neutrophils and PBMC) were separated from red blood cells (RBC) by differential sedimentation using 1.5% dextran in PBS. Neutrophils were separated from PBMC by Ficoll-Paque PLUS gradient

RNA isolation and RT-PCR
Total RNA was isolated by Trizol (Invitrogen, CA, USA) according to manufacturer's protocol. RNA (1 mg) was reversetranscribed to cDNA by ThermoScript RT-PCR system (Invitrogen, CA, USA). PCR was performed using Go Taq green master mix (Promega, USA). Primers for IL-1ra: forward primer: GGCCTCCGCAGTCACCTAATCACTCT, reverse primer: TACTACTCG TCCTCCTGGAAGTAGAA. The PCR conditions were as follows: 94uC for 1 minute followed by 35 cycles of 94uC for 1 minute, 58uC for 30 seconds, 72uC for 2 minutes and a final step of 72uC for 10 minutes.

Measurements of cytokines, chemokines and IL-1ra
To determine whether DH-PS changed the profiles of the secretions of cytokines and/or chemokines in vivo, mouse sera (obtained from facial vein blood sampling, Lancet) were collected at 0 (before the injection), 2 and 18 hrs after the intraperitoneal injection of DH-PS or PBS. Cytokines and chemokines were quantified by the Beadlyte mouse 21-Plex Cytokine Detection system (Millipore, Temecula, CA). For the detection of IL-1ra, sera were quantified by Mouse IL-1ra/IL-1F3 Quantikine ELISA Kit (

Validation of viability or the proliferation of THP-1 cells
Cell viability and proliferation assays were determined by adding MTS reagents to cell culture medium at 1:5 (v/v) (Promega, USA) and incubating for another 1-4 hr as the manufacturer's suggestions. The results were observed through the detection of the absorbance at 490 nm by spectrophotometer (Molecular Devices, USA).

Statistical analysis
The Student's t test was utilized to analyze the results of cytokine and chemokine productions, cell numbers of subpopulations of splenocytes, IL-1ra measurements, assays of the inhibitortreated cell viability and cell proliferation. For the determination of statistical significance of results in Figure 8, S1 and S2, the mean concentrations of IL1ra (pg/ml) (Fig.8) and mean values of absorbance at 490 nm (Fig.S1, S2) were used to statistical analysis before being converted to fold of control. P value was considered to be significant at , 0.05. Data were expressed as the mean values 6 standard deviation (S.D). Figure S1 Kinase inhibitors were not toxic to THP-1 cells in indicated concentrations. Cells were cultured (2610 6 cells/ml) with inhibitors for ERK/ELK (PD98059, 10 mM), JNK (SP600125, 1 mM), p38 MAPK (SB203580, 1 mM), PI3K (Ly294002, 10 mM), NFkB (Helenalin and MG132, 1 mM) or DMSO (0.1%) as control for 18 hrs. Viability was determined by MTS assay. Results were presented as fold of control (Y-axis) derived from the mean values of absorbance at 490 nm of inhibitor-treated groups divided by DMSO control group and error bars showed the standard deviation of triplicate.