Immunomodulatory activity of polysaccharides isolated from Clerodendrum splendens: beneficial effects in experimental autoimmune encephalomyelitis

Abstract

Background: Extracts of leaves from Clerodendrum have been used for centuries to treat a variety of medicinal problems in tropical Africa. However, little is known about the high-molecular weight active components conferring therapeutic properties to these extracts.

Methods: Polysaccharides from the leaves of Clerodendrum splendens were extracted and fractionated by ion exchange and size-exclusion chromatography. Molecular weight determination, sugar analysis, degree of methyl esterification, and other chemical characterization of the fractions were performed. Immunomodulatory activity of the fractions was evaluated by determining their ability to induce monocyte/macrophage nitric oxide (NO), cytokine production, and mitogen-activated protein kinase (MAPK) phosphorylation. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice, and severity of EAE was monitored in mice treated with intraperitoneal (i.p.) injections of the most active polysaccharide fraction. Lymph nodes (LN) and spleen were harvested, and levels of cytokines in supernatants from LN cells and splenocytes challenged with myelin oligodendrocyte glycoprotein peptide were determined.

Results: Fractions containing type II arabinogalactan had potent immunomodulatory activity. Specifically, the high-molecular weight sub-fraction CSP-AU1 (average of 38.5 kDa) induced NO and cytokine [interleukin (IL)-1α, -1β, -6, -10, tumor necrosis factor (TNF; designated previously as TNF-α), and granulocyte macrophage-colony stimulating factor (GM-CSF)] production by human peripheral blood mononuclear cells (PBMCs) and monocyte/macrophages. CSP-AU1-induced secretion of TNF was prevented by Toll-like receptor 4 (TLR4) antagonist LPS-RS, indicating a role for TLR4 signaling. Treatment with CSP-AU1 also induced phosphorylation of a number of MAPKs in human PBMC and activated AP-1/NF-κB. In vivo treatment of mice with CSP-AU1 and CSP-NU1 resulted in increased serum IL-6, IL-10, TNF, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α/CCL3, and MIP-1β/CCL4. CSP-AU1 treatment of mice with EAE (50 mg/kg, i.p., daily, 13 days) resulted in significantly reduced disease severity in this experimental model of multiple sclerosis. Levels of IL-13, TNF, interferon (IFN)-γ, IL-17, and GM-CSF were also significantly decreased, whereas transforming growth factor (TGF)-β was increased in LN cells from CSP-AU1-treated EAE mice.

Conclusions: Polysaccharide CSP-AU1 is a potent natural innate immunomodulator with a broad spectrum of agonist activity in vitro and immunosupressive properties after chronic administration in vivo.

Figure 1

Effect of Clerodendrum polysaccharide fractions on murine macrophage nitric oxide production. Murine J774.A1 macrophages were incubated for 24 hr with the indicated concentrations of CSP-NU (■, panel A), CSP-AU (□, panel A), CSP-NB (●, panel A), CSP-AB (○, panel A), CSP-AU1 (♦, panel B), CSP-AU2 (◊, panel B), CSP-NU1 (▲, panel C), CSP-NU2 (Δ, panel C), or 200 ng/ml LPS (, panel A). Values are the mean ± S.D. of triplicate samples from one experiment, which is representative of two independent experiments. Statistically significant differences (* p<0.05) between PBS-treated cells and cells treated with polysaccharide fractions or LPS are indicated.

Figure 2

Effect of Clerodendrum polysaccharide fractions on monocyte/macrophage TNF production. Murine J774.A1 macrophages (panels A-C), human MonoMac-6 cells (panels D-F), or PBMCs (panels D-F) were incubated for 24 hr with the indicated concentrations of CSP-NU (■, panels A, D, and G), CSP-AU (□, panels A, D, and G), CSP-NB (●, panels A, D, and G), CSP-AB (○, panels A, D, and G), CSP-AU1 (♦, panels B, E, and H), CSP-AU2 (◊, panels B, E, and H), CSP-NU1 (▲, panels C, F, and I), CSP-NU2 (Δ, panels C, F, and I), or 200 ng/ml LPS ( , panels A, D, and G). Cell-free supernatants were collected, and extracellular TNF was quantified by ELISA. Values are the mean ± S.D. of triplicate samples from one experiment, which is representative of three independent experiments. Statistically significant differences (* p<0.05) between PBS-treated cells and cells treated with polysaccharide fractions or LPS are indicated.

Figure 3

Effect of Clerodendrum polysaccharide fractions on macrophage IL-6 production. Murine J774.A1 macrophages were incubated for 24 hr with the indicated concentrations of CSP-NU (■, panel A), CSP-AU (□, panel A), CSP-NB (●, panel A), CSP-AB (○, panel A), CSP-AU1 (♦, panel B), CSP-AU2 (◊, panel B), CSP-NU1 (▲, panel C), CSP-NU2 (∆, panel C), or 200 ng/ml LPS (, panel A). Cell-free supernatants were collected, and extracellular IL-6 was quantified by ELISA. Values are the mean ± S.D. of triplicate samples from one experiment, which is representative of three independent experiments. Statistically significant differences (* p<0.05) between PBS-treated cells and cells treated with polysaccharide fractions or LPS are indicated.

Figure 4

Effect of Clerodendrum polysaccharide fractions on monocyte/macrophage GM-CSF production. Human MonoMac-6 cells (panels A-C) or PBMCs (panels D-F) were incubated for 24 hr with the indicated concentrations of CSP-NU (■, panels A and D), CSP-AU (□, panels A and D), CSP-NB (●, panels A and D), CSP-AB (○, panels A and D), CSP-AU1 (♦, panels B and E), CSP-AU2 (◊, panels B and E), CSP-NU1 (▲, panels C and F), CSP-NU2 (∆, panels C and F), or 200 ng/ml LPS (, panels A and D). Cell-free supernatants were collected, and extracellular TNF was quantified by ELISA. Values are the mean ± S.D. of triplicate samples from one experiment, which is representative of three independent experiments. Statistically significant differences (* p<0.05) between PBS-treated cells and cells treated with polysaccharide fractions or LPS are indicated.

Figure 5

Effect of Con A and LPS on GM-CSF production by human PBMCs. PBMCs were incubated for 24 hr with the indicated concentrations of Con A (○) or LPS (□). Cell-free supernatants were collected, and extracellular GM-CSF was quantified by ELISA. Values are the mean ± S.D. of triplicate samples from one experiment, which is representative of two independent experiments. Statistically significant differences (* p<0.05) between PBS-treated cells and cells treated with Con A or LPS are indicated.

Figure 6

Cytokine array analysis of PBMC cytokine production after treatment with Clerodendrum polysaccharide fractions CSP-AU1 and CSP-NU1. Human PBMCs were incubated for 24 hr with 250 μg/ml of CSP-AU1 (open bars) and CSP-NU1 (hatched bars), or 200 ng/ml of LPS (solid bars), and production of cytokines in the supernatants was evaluated using a MultiAnalyte ELISArray kit. Cytokine expression is shown as fold increase compared to background (PBS-treated cells). The data are presented as mean ± SD of duplicate samples from one experiment that is representative of two independent experiments. Statistically significant differences (* p<0.05) between PBS-treated cells and cells treated with polysaccharide fractions or LPS are indicated.

Figure 7

Effect of Clerodendrum polysaccharide fractions on AP-1/NF-κB activation. Human THP-1Blue monocytes were incubated for 24 hr with the indicated concentrations of CSP-NU (■, panel A), CSP-AU (□, panel A), CSP-NB (●, panel A), CSP-AB (○, panel A), CSP-AU1 (♦, panel B), CSP-AU2 (◊, panel B), CSP-NU1 (▲, panel C), CSP-NU2 (∆, panel C), or 200 ng/ml LPS (, panel A). Alkaline phosphatase activity was analyzed spectrophotometrically (absorbance at 655 nm) in the cell supernatants, as described. Values are the mean ± S.D. of triplicate samples from one experiment, which is representative of two independent experiments. Statistically significant differences (* p<0.05) between PBS-treated cells and cells treated with polysaccharide fractions or LPS are indicated.

Figure 8

Effect of TLR4 antagonist LPS-RS on TNF production by monocyte/macrophages activated with Clerodendrum polysaccharide fraction CSP-AU1. Human PBMCs (Panel A) or MonoMac-6 cells (Panel B) were pretreated with indicated concentrations LPS-RS for 30 min, followed by treatment with media (negative control, ●), CSP-AU1 (250 μg/ml, ○), or LPS (200 ng/ml, □) for 24 hr, and extracellular TNF was quantified by ELISA. Values are the mean ± S.D. of triplicate samples from one experiment, which is representative of three independent experiments. Statistically significant differences (* p<0.05) between PBS-treated cells and cells treated with the indicated concentrations of LPS-RS plus fraction CSP-AU1 or LPS are indicated.

Figure 9

Effect of CSP-AU1 on MAP kinase phosphorylation. PBMCs were incubated for 30 min with 250 μg/ml of CSP-AU1, and levels of kinase phosphorylation were evaluated using a phospho-MAPK array kit. The data are presented as mean ± SD of duplicate samples from one experiment. Statistically significant differences (* p<0.05) between PBS-treated cells and cells treated with CSP-AU1 are indicated.

Figure 10

Effect of Clerodendrum polysaccharide fractions on serum cytokines. Balb/c mice (n=4) were treated by i.p. injection with polysaccharide fractions CSP-AU1, CSP-AU2, CSP-NU1 (single dose of 250 mg/kg in 100 μl of PBS), LPS (single dose of 500 μg/kg in 100 μl of PBS), or PBS (100 μl). After 2 hr, mice were sacrificed, and serum was collected. Serum cytokine levels were evaluated using a MultiAnalyte ELISArray kit. Cytokine levels are shown as fold increase compared to background (PBS-treated mice). Inset A. CSP-AU1 was administrated i.p. (single dose 250 mg/kg), mice (n=3) were sacrificed at the indicated time points, and serum was collected. Inset B. The indicated doses of CSP-AU1 were administrated i.p., the mice (n=3) were sacrificed after 2 hr, and serum was collected. Serum TNF levels were evaluated using ELISA. For the Figure and Insets, the data are presented as mean ± S.D. of triplicate samples from different mice and are representative of two independent experiments. Statistically significant differences (* p<0.05) between animals treated with PBS and CSP-AU1 or LPS are indicated.

Figure 11

Effect of CSP-AU1 on EAE. C57BL/6 mice were treated i.p. daily with 50 mg/kg of CSP-AU1 (○) or PBS (●), starting on day 4 after immunization with MOG_35-55 peptide. The average score of 14 mice per group is shown, and statistically significant differences (* p<0.05) between EAE mice treated with PBS or fraction CSP-AU1 are indicated. A representative experiment of two independent experiments is shown.

Figure 12

Cytokine profile of splenic and lymph node (LN) cells isolated from PBS- or CSP AU1-treated EAE mice. Splenic and LN cells were prepared from mice at day 17 after immunization with MOG_35-55 peptide. These cells (5 × 10⁶ cell/ml) were restimulated with 10 μg/ml MOG_35-55 for 4 days, cell-free supernatants were collected, and extracellular cytokines were quantified by ELISA. Mean cytokine concentrations from 3–5 cultures ± S.D. are shown. Statistically significant differences (* p<0.05; ** p<0.001) between cells, isolated from PBS- and CSP-AU1-treated EAE mice are indicated.