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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

The aim of this study was to investigate whether the pectic polysaccharides BP-II, Oc50A1.I.A and CC1P1 isolated from the Malian medicinal plants Biophytum petersianum, Opilia celtidifolia and Cola cordifolia, respectively, were able to protect against Streptococcus pneumoniae infection in mice. The pectin preparations were administered intraperitoneally 3 h before challenge with S. pneumoniae serotype 6B. Blood samples were obtained from all animals before and at 3 h, 24 h and 72 h after challenge with the pneumococci. The number of bacteria in blood was recorded and the blood concentration of a range of cytokines measured. The pretreatment with BP-II, Oc50A1.I.A and CC1P1 demonstrated a protective activity against S. pneumoniae serotype 6B infection, albeit at different range of concentrations. The pectins showed no direct antibacterial effects towards S. pneumonia; however, they induced the production of a range of cytokines and chemokines. We have previously shown that BP-II, Oc50A1.I.A and CC1P1 exhibit complement fixation activity and also that BP-II and Oc50A1.I.A stimulate macrophages to produce NO. The observed clinical effect might therefore be linked to the ability of the pectic polysaccharides to stimulate the innate immune system.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

The gram-positive diplococcus Streptococcus pneumoniae is responsible for more than 50% of community-acquired cases of pneumonia, and an infection may develop into lethal diseases like septicaemia and meningitis [1]. Despite the advances in diagnostic methods and intensive care support, mortality among patients with pneumococcal pneumonia remains high, ranging from 5% to 35%, and there is also a growing antibiotics resistance among S. pneumoniae strains [2]. In Mali, West Africa, there is a limited access to western standard health care and children are not regularly immunized against S. pneumoniae. The case fatality rate due to invasive pneumococcal disease (IPD) in hospitalized children in Mali has been reported to be about 20% [3]. Worldwide, an estimated 1.6 million people, including 1 million children less than 5 years old, die of IPD annually [4]. Efforts to find good alternative preventive and curative agents are therefore of global interest.

One of the most promising recent alternatives to classical antibiotic treatment is the use of immunomodulators for enhancing host immune defence [5]. Polysaccharides such as β-glucans, inulin and pectins from various plants have attracted attention because of their effects on the immune system [6], in addition to their low toxicity and lack of harmful side effects [7]. Polysaccharides are easily obtained by extracting plant material with water at 50–100 °C, and as hot water extracts of plants are a common way to prepare traditional medicine, it is therefore relevant to study bioactivities of high-molecular-weight compounds in these extracts. Pectic polysaccharides with immunomodulating activities isolated from different Malian medicinal plants have been reported in several recent papers [8-10]. Although the medical effect of the pectic fractions' influence on the human immune system remains to be determined, our data suggest that they may participate in boosting the innate immune system in several ways such as through the complement system, by the influence on antigen-presenting cells like macrophages and dendritic cell (DC), and possibly by interacting with other molecules and cells in the immune system. Also, earlier reported analysis of cytokine secretion from purified rat B cells or DCs after stimulation with pectins isolated from the Malian medicinal plant Opilia celtidifolia Endl. Ex Walp. (Opiliaceae) suggests that the pectic polysaccharides may promote beneficial inflammation, since potent production of the pro-inflammatory cytokines IL-1α, IL-6 and TNF-α was observed [9]. However, B cells also produce high amounts of the anti-inflammatory cytokine IL-10 in vitro as response to the pectic polysaccharides, and thus, the pectins may also contribute to dampening or regulating the strength of the inflammatory processes [9].

Pectic polysaccharides are rich in galacturonic acid (GalA), and similar to other plant polysaccharides, pectins are polydisperse polymers, exhibiting significant heterogeneity with respect to both chemical structure and molecular weight. They consist of a number of structurally different regions, which include homogalacturonan (HG), rhamnogalacturonan I (RG-I) and substituted galacturonans, such as rhamnogalacturonan II (RG-II). The structural region RG-I may contain side chains of arabinogalactans type I (AG-I) or II (AG-II) [11-14].

The aim of the present paper was to study whether pectic polysaccharides isolated from Malian medicinal plants are able to protect against S. pneumoniae in a mouse infection model. We chose to test three pectic fractions, denominated BP-II, Oc50A1.I.A and CC1P1, which differ regarding their chemical composition and structure. BP-II was isolated from the aerial parts of Biophytum petersianum Klotzsch. (Oxalidaceae) [10], Oc50A1.I.A from the leaves of O. celtidifolia [9] and CC1P1 from the bark of Cola cordifolia (Cav.) R.Br. (Sterculiaceae) [15]. In Malian traditional medicine, B. petersianum is used in the treatment of wounds, inflammations, gastric ulcers, malaria and fever [16], O. celtidifolia is used against dermatitis, malaria, abdominal pain and as a wound healing remedy [9] and C. cordifolia in treating wounds, abdominal pain, malaria, infections and dysentery [15].

The present i.p. model for pneumococcal infection is well established as a model for systemic infection that gives reproducible bacteraemia [17]. S. pneumoniae serotype 6B was chosen for these studies. Serotype 6B is of intermediate virulence in mice and has a relatively protracted course of infection in the animals compared with certain other serotypes like type 4 [18]. Because lipopolysaccharide (LPS) is a possible contaminator of all biological materials, it was included as a control in our experiments.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

Isolation of BP-II

The aerial parts of B. petersianum were collected in Blendio, Mali, in November 2002. The plant was identified by the Department of Traditional Medicine (DMT), Bamako, Mali, and a voucher specimen is kept in the herbarium at DMT (Voucher No. 2653). The pectic polysaccharide fraction BP-II was prepared from the aerial parts of B. petersianum as described in a previous paper [10].

Isolation of Oc50A1.I.A

The leaves of O. celtidifolia were collected in Mali and identified by DMT. A voucher specimen is deposited in the herbarium at DMT (Voucher No. 2052 and 2477). The pectic fraction Oc50A1.I.A was isolated from the dried leaves of O. celtidifolia as previously described [9].

Isolation of CC1P1

The bark of C. cordifolia was collected in Kolokani, Mali in 2004. The plant material was identified by DMT, and a voucher specimen is deposited in the herbarium at DMT (Voucher No. 1331). The pectin-like polysaccharide CC1P1 was prepared from the bark of C. cordifolia as previously described [15].

Lipopolysaccharide

Escherichia coli 055:B5 endotoxin (lot # L2637) was purchased from Sigma-Aldrich, St. Louis, MO, USA.

Animals

The animal experiments were approved by the National Animal Research Committee and were performed in accordance with the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes (ETS 123). Female, inbred NIH mice (Harlan Laboratories, Blackthorn, England) were 6 weeks of age at arrival and were acclimatized for 1 week before entering the experiments. The animals were housed in type III cages (Techniplast, Buguggiate, Italy) inside a negative pressure isolator in a light temperature- and relative humidity-controlled environment with free access to municipal water and rodent chow.

Experimental procedure

Four sets of experiments were performed. In each experiment, there were four groups with six animals in each group (Table 1). For animal identification, each mouse was marked by punching holes at various positions in the ears. The pectic polysaccharides BP-II, Oc50A1.I.A, CC1P1 or LPS were diluted in phosphate-buffered saline (PBS) at concentrations given in Table 1 and sterile filtered using 0.2-μm filters (Millipore, Billerica, MA, USA). On day 0, 3 h before challenge with S. pneumoniae, the mice were injected i.p. with 0.4 ml at indicated concentrations of BP-II, Oc50A1.I.A, CC1P1, LPS or PBS. At time = 0 (T = 0), blood samples were obtained and the mice were injected i.p. with a nominal dose of 1.0 × 106 colony-forming units (CFU) of S. pneumoniae serotype 6B. Further blood samples were obtained at 3 h (T = 3), 24 h (T = 24) and 72 h (T = 72) after challenge with the pneumococci. Aliquots of the blood were seeded onto plates for quantification of CFU, and other aliquots were used for the measurement of cytokine concentration. All animals were inspected several times each day with a frequency determined by the severity of the observed clinical signs. The experiments were terminated at day 5 for Experiment 1 and at day 4 for Experiments 2–4.

Table 1. Experimental protocol for i.p. treatment of NIH mice with BP-II, Oc50A1.I.A, CC1P1 or LPS, 3 h before challenge with 1.0 × 10CFU S. pneumoniae serotype 6B on day 0. The experiments were terminated at day 5 for Experiment 1 and at day 4 for Experiments 2–4
 Treatment group ITreatment group IITreatment group IIITreatment group IV
Experiment 1

BP-II

(125 μg in 0.4 ml)

BP-II

(25 μg in 0.4 ml)

BP-II

(5 μg in 0.4 ml)

PBS

(0.4 ml)

Experiment 2

Oc50A1.I.A

(125 μg in 0.4 ml)

Oc50A1.I.A

(25 μg in 0.4 ml)

Oc50A1.I.A

(5 μg in 0.4 ml)

PBS

(0.4 ml)

Experiment 3

CC1P1

(100 μg in 0.4 ml)

CC1P1

(10 μg in 0.4 ml)

CC1P1

(1 μg in 0.4 ml)

PBS

(0.4 ml)

Experiment 4

LPS

(1000 ng in 0.4 ml)

LPS

(100 ng in 0.4 ml)

LPS

(10 ng in 0.4 ml)

PBS

(0.4 ml)

Determination of the clinical score

All animals were inspected and scored for clinical symptoms several times each day, using a study specific score sheet to identify adverse clinical effects and humane endpoints. The accumulated clinical score was based on the following predetermined clinical signs: body weight, piloerection, hunched posture, reduced level of activity, dehydration and altered respiration pattern. Each clinical sign was scored on a 0–4 scale, where 0 represented normality and 1–4 represented mild, moderate, marked and severe symptoms. An accumulated clinical score of more than 12 for all 6 clinical signs, a score of more than 7 for 2 clinical signs or weight loss above 15% within a 48-h period constituted the predetermined humane endpoint. Mice with an accumulated clinical score equal to or exceeding the predetermined humane endpoint maximum score were killed by cervical dislocation. When killed according to predetermined humane endpoint criteria, the outcome was recorded as death.

Mouse blood sampling

Blood samples (25 μl) were obtained from the saphenous vein as previously described [19].

Quantification of colony-forming units (CFU) in blood and challenge dose

Peripheral venous blood (10 μl) was serially diluted 10-fold in Todd–Hewitt broth, and 25 μl of four dilution steps was plated in triplicates on Columbia agar plates supplemented with 5% horse blood. The agar plates were incubated overnight at 35 °C, 5% CO2, and the colonies counted visually. The actual CFU injected in each of the four experiments was determined by diluting the inoculates 10-fold in Todd–Hewitt broth, and 100 μl of the theoretical 103, 102 and 101 concentrations was plated in triplicates onto Columbia agar plates supplemented with 5% horse blood. The agar plates were incubated overnight at 35 °C, 5% CO2, and the colonies counted visually.

Measurement of cytokine release in blood

Peripheral venous blood (10 μl) was diluted 10-fold in Todd–Hewitt broth, centrifuged to obtain plasma and stored at −70 °C until use. The levels of cytokines in plasma were quantified using the Bio-Plex ProTM Assays (Bio-Rad Laboratories, Hercules, CA, USA). The Bio-Plex cytokine assay is based on magnetic beads and is designed for the multiplexed quantitative measurement of multiple cytokines in a test sample in a single well. In our experiments, the premixed multiplex beads of the Bio-Plex Pro Mouse Cytokine 23-plex covering interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-γ), keratinocyte-derived chemokine (KC), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, RANTES and tumour necrosis factor (TNF)-α were used. The Bio-Plex Pro Mouse Cytokine Group II Assay was used for the detection of macrophage inhibitory protein 2 (MIP-2). The assays were performed as described by the manufacturer. In brief, the premixed standards were reconstituted in 200 μl standard diluent, and a serial 1:2 dilution in standard diluent was made to generate 8 points for the standard curve. The assays were performed in 96-well plates supplied with the assay kit. Premixed beads (50 μl) coated with target capture antibodies were transferred to each well of the plate and washed twice with Bio-Plex wash buffer. Duplicates of premixed standards or samples (50 μl) were added to each well containing washed beads. The plate was shaken for 30 s and then incubated at room temperature for 45 min with low-speed shaking. After incubation and washing, premixed detection antibodies (25 μl) were added to each well. The incubation was terminated after shaking for 45 min at room temperature. After three times washing, the beads were added streptavidin-PE (50 μl) and incubated at room temperature for 10 min with low-speed shaking. The plate was washed three times, and the beads resuspended in 125 μl of Bio-Plex assay buffer. Beads were read on the Bio-Plex suspension array system, and the data were analysed using Bio-Plex ManagerTM software with 5PL curve fitting.

Statistics

Nonparametric statistics on the quantification of CFU were performed with GraphPad Prism version 5.0 (GraphPad Software Inc., La Jolla, CA, USA). One-tailed Mann–Whitney U-test was used to compare the treatment groups. P values <0.05 were considered statistically significant. Statistics on dose–response relationships of cytokine release were performed with two-way ANOVA using IBM SPSS Statistics version 20. P values < 0.05 were considered statistically significant.

Limulus Amebocyte Lysate (LAL) assay

The LPS content in BP-II, Oc50A1.I.A, CC1P1 and PBS given i.p to the mice was determined using the Endpoint Chromogenic Limulus Amebocyte Lysate (LAL) test QCL-1000® (Lonza, Walkersville, MD, USA). The assay was performed according to the manufacturer's instructions. Briefly, a 1.0 EU/ml solution of endotoxin in LAL reagent water was prepared, and a serial dilution to generate four points for the standard curve was made. A 96-well microplate was pre-equilibrated at 37 °C, and while leaving the microplate at 37 °C, 50 μl of samples, standards or LAL reagent water was dispensed in duplicates in wells. Volumes of 50 μl of LAL were added to each well at time T = 0. At T = 10 min, 100 μl of chromogenic substrate preheated to 37 °C was added. Finally, at T = 16 min, 50 μl of stop reagent (25% v/v glacial acetic acid in water) was added to each well, and the absorbance was read at 415 nm.

Antibacterial activity

An antibacterial susceptibility test was carried out using the agar diffusion method. S. pneumoniae serotype 6B was streaked onto Petri dishes of Colombia agar supplemented with 5% horse blood and incubated overnight at 35 °C, 5% CO2. Inocula of the micro-organism were prepared by suspending the bacteria in 2 ml of Mueller–Hinton broth until the absorbance of the suspension corresponded to McFarland standard no. 5. Petri dishes of Mueller–Hinton supplemented with 5% horse blood were aseptically inoculated with the test organisms using sterile Pasteur pipette and excess inoculum removed. The plates were allowed to dry for 10–15 min. Each Petri dish was divided into five sectors for BP-II, Oc50A1.I.A and LPS and three sectors for CC1P1. In each sector, an 8-mm bore was made using a flamed, but cooled, borer. Three different concentrations of BP-II (3, 0.3 and 0.03 mg/ml), Oc50A1.I.A (3, 0.3 and 0.03 mg/ml), LPS (0.4, 0.04 and 4 μg/ml) or 1 mg/ml of CC1P1 were used. As a positive control, 0.06 mg/ml of gentamicin sulphate (Sigma) was used. Mueller–Hinton broth was used as negative control. Volumes of 180 μl stock solution of the pectic extracts, LPS, positive and negative control were loaded into separate bores. The plates were left in room temperature for 25 min before incubation overnight at 35 °C, 5% CO2. After a 22-h incubation period, the zone of inhibitions was measured to the nearest millimetre. The presence of a zone of inhibition was regarded as the presence of antimicrobial activity. The experiments were performed in three replicates.

Bacteria

A strain of Streptococcus pneumoniae serotype 6B was kindly supplied by Dr. Jan Poolman, RIVM, The Netherlands. It was kept frozen and prepared for challenge as described [17].

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

Bacteraemia and clinical score in BP-II-treated mice

Three hours after challenge with 0.9 × 10CFU S. pneumoniae, there was a statistically significant difference in the bacteraemia levels in the mice treated with 25 μg BP-II or 125 μg BP-II (< 0.01), compared to the PBS-treated mice (Fig. 1A, Table 2). Among the BP-II treated mice, the mice given a dose of 125 μg (< 0.05) or 25 μg (< 0.01) had statistically significant lower levels of bacteraemia than the mice given 5 μg 3 h after pneumococcal challenge (Table 2). At T = 24, the levels of CFU had decreased for both the BP-II- and the PBS-treated mice, but the bacteraemia in the mice given 5 μg, 25 μg or 125 μg BP-II were statistically significant lower (< 0.01) compared to the PBS-treated mice (Fig. 1A, Table 2). No statistically significant difference in bacteraemia between the mice pretreated with BP-II or PBS was observed 72 h after challenge with Spneumoniae. Comparing the different doses of BP-II given, no statistically significant differences in bacteraemia were observed 24 h after pneumococcal challenge, while at T = 72, the mice given 125 μg (< 0.05) had statistically significant lower levels of bacteraemia than the mice given 5 μg.

Table 2. Bacteraemia (CFU median numbers) of female NIH mice infected i.p. with pneumococci serotype 6B after pretreatment with BP-II, Oc50A1.I.A, CC1P1 or LPS. ND: no data (group terminated prior to sampling). The results are expressed with 95% confidence intervals in brackets
Hours after challenge
PretreatmentChallenge dose (×104)32472
CFU × 104 (95% CI)
  1. a

    Lower levels of bacteraemia in BP-II 125 μg (< 0.05) and BP-II 25 μg (< 0.01) compared to BP-II 5 μg.

  2. b

    Lower levels of bacteraemia in BP-II 125 μg (< 0.05) compared to BP-II 5 μg.

  3. c

    Lower levels of bacteraemia in Oc50A.1.I.A 25 μg (< 0.05) and Oc50A.1.I.A 5 μg (< 0.01) compared to Oc50A.1.I.A 125 μg.

  4. d

    Lower levels of bacteraemia in CC1P1 100 μg (< 0.01) compared to CC1P1 10 μg and 1 μg.

  5. e

    Lower levels of bacteraemia in CC1P1 10 μg (< 0.05) compared to CC1P1 1 μg.

  6. f

    Lower levels of bacteraemia in CC1P1 100 μg and 10 μg (< 0.01) compared to CC1P1 1 μg.

  7. g

    Lower levels of bacteraemia in LPS 1000 ng (< 0.05) compared to LPS 100 ng and 10 ng.

BP-II 125 μg90150 (70, 290)a0.4 (−0.07, 1.6)0.3 (−0.1, 1.5)b
BP-II 25 μg9040 (8, 100)a0.2 (−0.1, 0.9)1.0 (−4.1, 12)
BP-II 5 μg90300 (190, 370)0.8 (0.3, 1.4)1.9 (−22, 55)
Saline (PBS)90540 (270, 850)3.9 (1.7, 8.0)1.1 (−0.6, 5.7)
Oc50A1.IA 125 μg110290 (170, 400)1.4 (0.2, 2.9)1.5 (−0.6, 5.7)
Oc50A1.IA 25 μg110130 (70, 200)c3.8 (−0.9, 11)0.4 (−0.4, 2.8)
Oc50A1.IA 5 μg110160 (70, 250)c0.2 (−0.04, 0.8)0.7 (0.05, 1.5)
Saline (PBS)1101000 (390, 1700)20 (1, 55)2.8 (−16, 22)
CC1P1 100 μg10592 (2.2, 250)d0.5 (−0.08, 1.3)f0.2 (−4.8, 20)
CC1P1 10 μg105420 (300, 560)e0.4 (−7.2, 20)f1.2 (−0.02, 2.1)
CC1P1 1 μg105940 (460, 1300)61 (18, 76)ND
Saline (PBS)105500 (240, 660)3.8 (−28, 66)0.6 (−1.5, 4.1)
LPS 1000 ng10670 (40, 140)0.1 (0.06, 0.2)g0.2 (−17, 41)
LPS 100 ng10660 (20, 140)0.2 (−0.4, 1.4)0.7 (−2.3, 7.9)
LPS 10 ng10680 (20, 140)1.1 (−2.4, 8.0)0.08 (−0.05, 0.4)
Saline (PBS)106410 (270, 550)4.5 (0.3, 1.3)1.7 (−2.5, 15)
image

Figure 1. (A) Colony-forming units (CFU) in peripheral blood from female NIH mice pretreated with the pectin BP-II or saline (PBS) on day 0 (see Table 1). The mice were pretreated with the test substance 3 h before challenge with 0.9 × 106 pneumococci 6B at T = 0. The median for each treatment group is marked with a vertical line. Non-parametric statistics were performed, and one-tailed Mann-Whitney was used to compare the treatment groups. P values < 0.05 were considered statistically significant. (B) Sum up of clinical score observed in cages given BP-II or PBS. Three out of six mice given 5 μg BP-II and four out of six PBS-treated mice did in addition develop signs of otitis interna fours days after inoculation with S. pneumoniae. Data points are mean numbers of clinical score of six mice.

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The mice pretreated with 125 μg BP-II showed transient signs of discomfort, reduced activity and piloerection just after injecting the pectin. This was not observed for the mice given 5 μg BP-II, 25 μg BP-II or PBS (Fig. 1B). The symptoms lasted only for a short period, and at the time of bacterial challenge, the mice looked normal. After challenge with S. pneumoniae, the mice pretreated with PBS had higher clinical scores during the whole experiment compared to the mice given BP-II (Fig. 1B). The clinical signs of infection started approximately 8 h after challenge, the main symptoms being piloerection, closed eyes and a reduced level of activity. Three of six mice given 5 μg BP-II and four of six PBS-treated mice developed symptoms of otitis interna, demonstrated by head tilt and disturbed sense of balance, 4 days after inoculation with S. pneumoniae. The mice given 125 μg BP-II or 25 μg BP-II showed no symptoms of infection and remained active and normal. A dose of minimum 25 μg BP-II seems to be necessary to protect against pneumococcal infection and at the same time reducing the risk of otitis interna.

Bacteraemia and clinical score in Oc50A1.I.A-treated mice

Three hours after challenge with 1.1 × 106 CFU of Spneumoniae, there was a statistically significant difference in the levels of bacteraemia in the mice treated with 5 μg Oc50A1.I.A, 25 μg Oc50A1.I.A or 125 μg Oc50A1.I.A (< 0.01), compared to the PBS-treated mice (Fig. 2A, Table 2). Among the Oc50A1.I.A-treated mice, the mice given a dose of 25 μg (< 0.05) or 5 μg (< 0.01) had statistically significant lower levels of bacteraemia than the mice given 125 μg 3 h after pneumococcal challenge. At T = 24, the levels of CFU had decreased for both the Oc50A1.I.A- and the PBS-treated mice, but the bacteraemia in the mice given 5 μg (< 0.01), 25 μg (< 0.05) or 125 μg (< 0.01) Oc50A1.I.A were statistically significant lower compared to the PBS-treated mice (Fig. 2A, Table 2). There was no statistically significant difference in the levels of bacteraemia between the Oc50A1.I.A- and the PBS-treated mice at T = 72. Comparing the different doses of Oc50A1.I.A given, no statistically significant differences in bacteraemia were observed 24 h or 72 h after pneumococcal challenge.

image

Figure 2. (A) Colony-forming units (CFU) in peripheral blood from female NIH mice pretreated with the pectin Oc50A1.I.A or saline (PBS) on day 0 (see Table 1). The mice were pretreated with the test substance 3 h before challenge with 1.1 × 106 pneumococci 6B at T = 0. The median for each treatment group is marked with a vertical line. Non-parametric statistics were performed, and one-tailed Mann-Whitney was used to compare the treatment groups. P values < 0.05 were considered statistically significant. (B) Sum up of clinical score observed in cages given Oc50A1.I.A or PBS. Three of the mice in the PBS-group were euthanized at day 1, and one at day 2. The surviving mice given PBS developed signs of otitis interna 3 days after inoculation with S. pneumoniae. Data points are mean numbers of clinical score of six mice.

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After pretreatment with pectin and before challenge with Spneumoniae, the mice given 25 μg or 125 μg Oc50A1.I.A showed transient signs of discomfort with a lower degree of activity and piloerection. These symptoms lasted only for a short period, and at the time of bacterial challenge, the mice looked normal. After challenge with Spneumoniae, the mice pretreated with PBS had higher clinical scores during the whole experiment (Fig 2B). The signs of infection started approximately 8 h after challenge, the main symptoms being a lower degree of activity, closed eyes, piloerection and increased/laboured respiration. Three of the mice in the PBS group were euthanized on day 1, and 1 on day 2. The animal euthanized at day 2 showed signs of otitis interna. The mice given 5 μg Oc50A1.I.A, 25 μg Oc50A1.I.A or 125 μg Oc50A1.I.A showed no symptoms of infection and remained active and normal. A dose of minimum 5 μg Oc50A1.I.A seems to be necessary to protect against pneumococcal infection and at the same time reducing the risk of otitis interna.

Bacteraemia and clinical score in CC1P1-treated mice

Three hours after challenge with 1.05 × 10CFU of Spneumoniae, there was a statistically significant difference in the levels of bacteraemia in the mice treated with 100 μg CC1P1 (< 0.01) compared to the PBS-treated mice (Fig. 3A, Table 2). Among the CC1P1-treated mice, the mice given a dose of 100 μg had statistically significant lower levels of bacteraemia than the mice given 10 μg or 1 μg (< 0.01) 3 h after pneumococcal challenge, while a dose of 10 μg (< 0.05) gave a statistically significant lower bacteraemia than the mice given 1 μg. No statistically significant difference in the bacteraemia levels in the mice treated with CC1P1 compared with the PBS-treated mice was observed 24 h or 72 h after pneumococcal challenge. Comparing the different doses of CC1P1 given, the mice given 100 μg or 10 μg (< 0.01) had statistically significant lower levels of bacteraemia than the mice given 1 μg at T = 24, while no statistically significant differences in bacteraemia were observed at T = 72.

image

Figure 3. (A) Colony-forming units (CFU) in peripheral blood from female NIH mice pretreated with the pectin CC1P1 or saline (PBS) on day 0 (see Table 1). The mice were pretreated with the test substance 3 h before challenge with 1.05 × 106 pneumococci 6B at T = 0. The median for each treatment group is marked with a vertical line. Non-parametric statistics were performed, and one-tailed Mann-Whitney was used to compare the treatment groups. P values < 0.05 were considered statistically significant. (B) Sum up of clinical score observed in cages given CC1P1 or PBS. Two of the mice in the PBS-group were euthanized at day 1. All six of the mice given 1 μg CC1P1 were euthanized at day 1. At day 2 one of the mice given 10 μg CC1P1 were found dead. Surviving mice given PBS developed signs of otitis interna 3 days after inoculation with S. pneumoniae. Data points are mean numbers of clinical score of six mice.

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After challenge with Spneumoniae, the mice pretreated with 1 μg CC1P1 or PBS had higher clinical scores compared to the mice given 10 μg or 100 μg of CC1P1 (Fig 3B). The signs of infection started approximately 8 h after challenge, the main symptoms being a lower degree of activity, closed eyes, piloerection and increased/laboured respiration. Two of the mice in the PBS group were euthanized at day 1, one of the mice showing signs of otitis interna. All six of the mice given 1 μg CC1P1 were euthanized at day 1. At day 2, one of the mice given 10 μg CC1P1 was found dead between inspection intervals. The mice given 100 μg CC1P1 showed no symptoms of infection and remained active and normal, while the surviving animals in the group given 10 μg CC1P1 or PBS recovered and showed no symptoms from day 2. A dose of minimum 100 μg CC1P1 seems to be necessary to protect against pneumococcal infection and at the same time reducing the risk of otitis interna.

Bacteraemia and clinical score in LPS-treated mice

Three hours after challenge with 1.06 × 106 CFU of Spneumoniae, there was a statistically significant difference in the bacteraemia levels in the mice treated with 10 ng, 100 ng or 1000 ng LPS (< 0.01) compared with the PBS-treated mice (Fig. 4A, Table 2). Among the LPS-treated mice, no statistically significant differences in bacteraemia were observed 3 h after pneumococcal challenge. At T = 24, the levels of CFU had decreased in both the LPS- and the PBS-treated mice, but the bacteraemia in the mice given 10 ng (< 0.05), 100 ng (< 0.01) or 1000 ng (< 0.01) LPS were statistically significant lower compared to the PBS-treated mice (Fig. 4A, Table 2). Seventy-two hours after challenge with Spneumoniae, only 10 ng LPS (< 0.05) gave a statistically significant difference in bacteraemia compared to PBS. Comparing the different doses of LPS given, the mice given 1000 ng (< 0.05) had statistically significant lower levels of bacteraemia than the mice given 100 ng or 10 ng at T = 24, while the mice given 10 ng (< 0.05) had statistically significant lower levels of bacteraemia than the mice given 100 ng at T = 72.

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Figure 4. (A) Colony-forming units (CFU) in peripheral blood from female NIH mice pretreated with LPS or saline (PBS) on day 0 (see Table 1). The mice were pretreated with the test substance 3 h before challenge with 1.06 × 106 pneumococci 6B at T = 0. The median for each treatment group is marked with a vertical line. Non-parametric statistics were performed, and one-tailed Mann-Whitney was used to compare the treatment groups. P values < 0.05 were considered statistically significant. (B) Sum up of clinical score observed in cages given LPS or PBS. One of the mice in the PBS-group developed symptoms of otitis interna 3 days after inoculation with S. pneumoniae. Data points are mean numbers of clinical score of six mice.

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After challenge with Spneumoniae, the mice pretreated with PBS had higher clinical scores during the whole experiment (Fig 4B). The signs of infection started approximately 8 h after challenge, the main symptoms being a lower degree of activity, closed eyes, piloerection, hunched posture and increased/laboured respiration. In addition, one of the mice in the PBS group developed symptoms of otitis interna 3 days after inoculation with S. pneumoniae. The mice given LPS showed weak or no symptoms of infection and remained active and normal throughout the experiment. A dose of minimum 10 ng LPS seems to be necessary to protect against pneumococcal infection and at the same time reducing the risk of otitis interna.

Plasma levels of cytokines in BP-II treated mice

At T = 0, 3 h after i.p. prechallenge with BP-II, statistically significant dose–response relationships in the release of IL-1α, IL-6, MCP-1, MIP-2, G-CSF, KC (Fig. 5), IL-1β, IL-9, IL-12 (p40) (Figure S1), IL-13, MIP-1α and RANTES (Figure S2) in plasma were observed. Three hours after challenge with S. pneumoniae (T = 3), statistically significant dose–responses were observed in the plasma levels of IL-1α, MCP-1, MIP-2, G-CSF, KC (Fig. 5), IL-1β, IL-3, IL-4, IL-12 (p40), IL-12 (p70) (Figure S1), IFN-γ, MIP-1α, MIP-1β, RANTES and eotaxin (Figure S2). At T = 24, statistically significant dose–responses in the plasma levels of IL-2, IL-10, IL-13, TNF-α, MIP-1β and GM-CSF were observed (Figure S1 and S2), while at T = 72, only IL-9 and IL-17 gave significant dose–responses.

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Figure 5. Levels of IL-1α, IL-6, MCP-1, MIP-2, G-CSF and KC in mouse plasma from female NIH mice pretreated with BP-II or saline (PBS) (see Table 1). The mice were pretreated with the test substance 3 h before challenge with pneumococci 6B at T = 0. Data are mean cytokine concentrations with SD. Statistics on dose-response relationships of cytokine release were performed with Two-way ANOVA. Statistically significant dose-response relationships are indicated where present. P values < 0.05 were considered statistically significant.

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Pretreatment with BP-II gave a particularly strong induction in the levels of IL-1α, IL-6, MCP-1, MIP-2, G-CSF and KC, compared to the mice pretreated with PBS. Peak plasma levels of IL-6, MCP-1, MIP-2 and KC were observed at T = 0 in the mice given 125 μg BP-II, while peak levels of IL-1α and G-CSF were seen at T = 3 (Fig. 5). In the PBS-treated mice, the plasma levels of the cytokines IL-1α, MCP-1, MIP-2, G-CSF and KC (Fig. 5) increased sharply at T = 24 and were higher compared to the levels in the mice given BP-II. These cytokines have pro-inflammatory effects, and the high levels observed in the PBS-treated mice at T = 24 indicate a very active inflammatory response, which is also indicated by Fig. 1B.

Plasma levels of cytokines in Oc50A1.I.A-treated mice

At T = 0, 3 h after i.p. prechallenge with Oc50A1.I.A, statistically significant dose–response relationships in the release of IL-1α, IL-6, MCP-1, MIP-2, G-CSF, KC (Fig. 6), IL-9, IL-12 (p40) (Figure S3), IL-13, MIP-1α and RANTES (Figure S4) in plasma were observed. Three hours after challenge with S. pneumoniae (T = 3), statistically significant dose–responses were observed in the plasma levels of IL-1α, MCP-1, MIP-2, G-CSF, KC (Fig. 6), IL-1β, IL-2, IL-9 (Figure S3), IL-13, TNF-α, IFN-γ, MIP-1α, MIP-1β and GM-CSF (Figure S4).

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Figure 6. Levels of IL-1α, IL-6, MCP-1, MIP-2, G-CSF and KC in mouse plasma from female NIH mice pretreated with Oc50A1.I.A or saline (PBS) (see Table 1). The mice were pretreated with the test substance 3 h before challenge with pneumococci 6B at T = 0. Data are mean cytokine concentrations with SD. Statistics on dose-response relationships of cytokine release were performed with Two-way ANOVA. Statistically significant dose-response relationships are indicated where present. P values < 0.05 were considered statistically significant.

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Pretreatment with Oc50A1.I.A gave a particularly strong induction in the levels of IL-1α, IL-6, MCP-1, MIP-2, G-CSF and KC (Fig. 6), compared to the mice pretreated with PBS. Peak plasma levels of IL-1α, IL-6, MCP-1, MIP-2 and KC were observed at T = 0 in the mice given 125 μg Oc50A1.I.A, while the peak plasma level of G-CSF was observed at T = 3.

Plasma levels of cytokines in CC1P1-treated mice

At T = 0, 3 h after i.p. prechallenge with CC1P1, statistically significant dose–response relationships in the release of IL-1α, IL-6, MCP-1, MIP-2, G-CSF, KC (Fig. 7), IL-1β and IL-12 (p40) (Figure S5) in plasma were observed. Three hours after challenge with S. pneumoniae (T = 3), statistically significant dose–responses were observed in the plasma levels of IL-1β, IL-3, IL-4, IL-10, IL-12 (p70) (Figure S5), IL-17, MIP-1α, MIP-1β and GM-CSF (Figure S6), while at T = 24, only IL-1β gave a significant dose–response.

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Figure 7. Levels of IL-1α, IL-6, MCP-1, MIP-2, G-CSF and KC in mouse plasma from female NIH mice pretreated with CC1P1 or saline (PBS) (see Table 1). The mice were pretreated with the test substance 3 h before challenge with pneumococci 6B at T = 0. Data are mean cytokine concentrations with SD. Statistics on dose-response relationships of cytokine release were performed with Two-way ANOVA. Statistically significant dose-response relationships are indicated where present. P values < 0.05 were considered statistically significant.

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Pretreatment with CC1P1 particularly induced the levels of IL-1α, IL-6, MCP-1, MIP-2, G-CSF and KC (Fig. 7), compared to the mice pretreated with PBS. Peak plasma levels were observed at T = 0 for IL-1α, MCP-1, MIP-2 and KC, at T = 3 for IL-6 and at T = 24 for G-CSF in the mice given 100 μg CC1P1 (Fig. 7). In the mice given 1 μg CC1P1, the plasma levels of a range of pro-inflammatory cytokines increased sharply at T = 24. This indicates a very active inflammatory response, which is also indicated in Fig. 3B. High levels of pro-inflammatory cytokines were also observed in the PBS-treated mice, at T = 3 and T = 24 (Fig. 7).

Plasma levels of cytokines in LPS-treated mice

At T = 0, 3 h after i.p. prechallenge with LPS, statistically significant dose–response relationships in the release of IL-1α, IL-6, MCP-1, MIP-2, G-CSF, KC (Fig. 8), IL-1β, IL-12 (p40) (Figure S7), IL-13, MIP-1α, MIP-1β, RANTES and eotaxin (Figure S8) in plasma were observed. Three hours after challenge with S. pneumoniae (T = 3), statistically significant dose–responses were observed in the plasma levels of IL-1α, MCP-1, MIP-2, G-CSF, KC (Fig. 8), MIP-1α and RANTES (Figure S8).

image

Figure 8. Levels of IL-1α, IL-6, MCP-1, MIP-2, G-CSF and KC in mouse plasma from female NIH mice pretreated with LPS or saline (PBS) (see Table 1). The mice were pretreated with the test substance 3 h before challenge with pneumococci 6B at T = 0. Data are mean cytokine concentrations with SD. Statistics on dose-response relationships of cytokine release were performed with Two-way ANOVA. Statistically significant dose-response relationships are indicated where present. P values < 0.05 were considered statistically significant.

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Pretreatment with LPS gave a particularly strong induction in the levels of IL-1α, IL-6, MCP-1, MIP-2, G-CSF and KC, compared to the mice pretreated with PBS. Peak plasma levels of IL-1α, IL-6, MCP-1 and KC (Fig. 8) were observed at T = 0 in the mice given 1000 ng LPS, while peak levels of MIP-2 and G-CSF (Fig. 8) were seen at T = 3. The plasma levels of the cytokines IL-1α and G-CSF (Fig. 8) increased sharply in the PBS-treated mice at T = 24 and were higher compared to the levels in the mice given LPS. The pro-inflammatory effects of these cytokines indicate a very active inflammatory response in the PBS-treated mice at T = 24, which is also indicated in Fig. 4B.

Determination of LPS contamination in BP-II, Oc50A1.I.A, CC1P1 and PBS

Because LPS is a known immunomodulator and often contaminates biological preparations, the level of LPS contamination in BP-II, Oc50A1.I.A and CC1P1 was determined using the Limulus Amebocyte Lysate (LAL) Pyrogen test. The test was performed on the solutions given i.p. to the animals. The LPS content of the 5, 25 and 125 μg BP-II formulations was 15.5, 96.2 and 96.2 ng, respectively. The LPS content of the 5, 25 and 125 μg Oc50A1.I.A formulations was 104.8, 101.8 and 96.2 ng, respectively. The LPS content of the 1, 10 and 100 μg CC1P1 formulations was 1.5, 12.1 and 92.6 ng, respectively. The LPS content in the PBS given i.p. to the mice was also determined and shown to be non-existent.

Antibacterial activity of BP-II, Oc50A1.I.A, CC1P1 and LPS against S. pneumoniae

The direct antibacterial activity of the pectic polysaccharides and LPS against S. pneumoniae was tested using the agar diffusion method. The presence of a zone of inhibition after a 22-h incubation period was regarded as the presence of antimicrobial activity. No zone of inhibition was observed for the pectic extracts BP-II, Oc50A1.I.A and CC1P1 or from LPS. The positive control gentamicin sulphate (10.8 μg/bore) showed an inhibitor zone of approximately 14 mm in all the experiments. The experiments were performed in three replicates.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

The model for pneumococcal infection used in the present study gives reproducible bacteraemia [17]. By utilizing this mouse model, the pectic polysaccharides BP-II and Oc50A1.I.A isolated from the Malian medicinal plants B. petersianum and O. celtidifolia, respectively, protected against S. pneumoniae serotype 6B infection when administered i.p. 3 h before challenge with the bacteria, while CC1P1 isolated from C. cordifolia showed marginal protective effect. The protective effect was measured by a lower concentration of bacteria circulating in the blood and by a lower clinical scoring of predetermined clinical signs, compared to the mice pretreated with PBS only (Table 2, Fig. 1–3). The pectins also seemed to give a protection against pneumococcus-induced otitis interna, which occurred in some of the mice given PBS and 5 μg of BP-II. The lowest doses of the pectins giving significant protective effect were 25 μg/mouse of BP-II, 5 μg/mouse of Oc50A1.I.A and 100 μg/mouse of CC1P1 (Table 2, Fig. 1–3). To draw definite conclusions, the experiments should ideally have been reproduced. This was, however, made impossible due to a time-consuming and demanding experimental procedure, high costs and animal ethical issues.

Cytokines and chemokines are important signal and effector molecules in both the innate and adaptive immune systems. Furthermore, they mediate crosstalk and cooperation between the two compartments of the immune system. In our mouse infection model, two stages of cytokine/chemokine responses were anticipated, one when the pectin is injected and one at the following live pneumococci challenge. The quality and quantity of the cytokine/chemokine response at the two time points and in the cause of infection in relation to bacteraemia and clinical signs of the infection will shed light on protection mechanisms and immune responses induced by the pectins. The cytokine profile induced by the pectin prior to pneumococcal challenge would be of special interest. A time window of 3 h for cytokine induction of the pectins will naturally limit the observations to the easily released or rapidly recruited cytokines/chemokines and to the ones with high concentration in peripheral blood. With these limitations in mind, we observed striking dose-dependent responses for IL-1α, IL-6, MCP, MIP-2, G-CSF and KC compared to the response due to bacteria alone (the PBS response) before pneumococcal challenge (Fig. 5–7). Oc50A1.I.A elicited the strongest responses.

In a previous study, the native fraction of Oc50A1.I.A, Oc50A1.I.pur, was found to stimulate DCs to release IL-1α and IL-6 [9], while BP-II has been reported to increase the production of IL-6 from Peyer's patch cells [10]. The role of IL-1α is through its ability to facilitate infiltration of leucocytes to the inflamed site and activate them to secrete a cascade of inflammatory mediators that propagate and sustain the inflammatory response and is thought to contribute in protection against S. pneumoniae [20]. The interleukin IL-6 has been assigned both pro- and anti-inflammatory properties during the years and appears to be critical for the effective management of acute inflammation [21]. Interestingly, the mice that responded with the strongest induction of IL-1α and IL-6 before pneumococcal challenge were most protected against infection. The levels of IL-6 were shown to increase progressively in the sicker animals and correlated with the presence of bacteraemia, this was also the case in a mouse pneumococcal model described by Bergeron and co-workers [22].

Chemokines are essential for the recruitment of neutrophils during bacterial infections. An increase in MCP-1 levels in humans has been shown to mediate the initial steps of inflammation by recruiting leucocytes [23, 24]. The glutamic acid–leucine–arginine (ELR)-positive subgroup of CXC chemokines, like MIP-2 and KC, induces neutrophil chemotaxis [25], KC being the murine equivalent to human IL-8 [26]. In the present study, the pretreatment with the pectic fractions led to an induction in the plasma levels of these three chemokines in plasma before pneumococcal challenge in a dose-dependent manner. The mice in which pretreatment with pectin induced the release of these cytokines before pneumococcal challenge were not severely affected by the infection. The opposite was observed in the mice where an induction of chemokine concentration before challenge was not observed.

Granulocyte colony-stimulating factor stimulates neutrophil proliferation and recruitment, and elevated local and systemic concentrations of G-CSF are found in patients with infections. This strongly suggests that G-CSF plays an important role in the elimination of invading organisms [1, 26]. Signs of otitis interna were observed in several of the PBS-treated mice (Fig. 1–4), but not in the mice where G-CSF levels were induced by the pectins before pneumococcal challenge. This may be in accordance with the results reported by [26, 27], showing that pretreatment with G-CSF reduces the risk of fatality and protects from cerebral damage after pneumococcal challenge.

Lipopolysaccharide has earlier been reported to increase the survival in mice infected with S. pneumoniae. In a study performed by Dallaire and co-workers [28], LPS was injected in concomitance with the inoculum, suggesting that an enhanced inflammatory response due to LPS in the early hours of infection might be beneficial. In the present study, LPS doses of 10–1000 ng/mouse gave a protection against pneumococcal infection given prechallenge. The LPS contamination of our protective pectic fractions was determined by the LAL test to be in the range 12.1–104.8 ng and possibly could explain the observed protective effect. The measured LPS levels in the pectins could, however, be due a false-positive result. The LAL test is based on the clotting response of horseshoe crab (Limulus polyphemus) blood upon contact with endotoxin. A number of polysaccharides react in the limulus assay via their ability to bind factor C, the first enzyme in the endotoxin-induced coagulation cascade in horseshoe crab blood [29, 30]. It is also possible that the effects of the putative LPS contamination could be additive or synergistic with an effect from the pectins. In a study showing a protective effect from the pectin PM-II, it was concluded that the pectin had an effect per se [18].

The putative LPS contamination of ≤105 ng in the injected pectic fractions could, however, not be responsible for the observed cytokine induction by 125 μg BP-II and all concentration ranges of Oc50A1.I.A. An LPS concentration of 1000 ng/mouse was necessary to induce a comparable amount of the cytokines IL-1α, IL-6, MCP, MIP-2, G-CSF and KC (Fig. 5–8). The observed pectin-cytokine dose–response, in the face of rather similar concentrations of LPS in the BP-II and Oc50A1.I.A formulations, also indicates that the effect of the putative LPS contamination is secondary to that of the pectins.

A considerable difference in cytokine responses among the three pectins was observed. This together with great difference in clinical course and in bacteraemia indicates differences in protective mechanism and protective efficacy between the pectins. Macrophages have for a long time been considered as one of the main target cells for polysaccharide interaction, and several compounds have been shown to modulate their cytokine production and to enhance phagocytic activity [31]. BP-II [8] and Oc50A1.I.A [9] have been reported to activate macrophages, and the observed induction in cytokine release by the pectic fractions means that they might be able to boost the early responses by macrophages.

Macrophage activation by plant polysaccharides is thought to be mediated primarily through the recognition of polysaccharide polymers by specific receptors. Toll-like receptor 4 (TLR4) has been identified as the main pattern recognition receptor expressed by macrophages and mediates macrophage activation by transmitting a variety of extracellular signals, resulting in the secretion of cytokines such as NO and TNF-α. An acidic polysaccharide isolated from the roots of Angelica sinensis (Oliv.) Diels has earlier been reported to promote NO production by upregulating the expression of TLR4 on macrophages [31]. The ability of the pectic polysaccharides BP-II, Oc50A1.I.A and CC1P1 to stimulate TLRs is, however, unknown.

In addition to TLR-4, macrophages might bind plant polysaccharides via CD14, complement receptor 3 (CR3), dectin-1 and mannose receptor. An activation of all these receptors leads to intracellular signalling cascades, resulting in transcriptional activation and the production of pro-inflammatory cytokines [30]. However, BP-II, Oc50A1.I.A and CC1P1 contain minor amounts of mannose residues and probably do not bind to the cellular mannose receptor. They contain small amounts of glucose, but this is considered to be contaminants. It is therefore unlikely that the pectins function by binding to the lectin site in CR3 on leucocytes similar to β-glucan, which has also shown to be protective in this pneumococcal infection model [32].

BP-II [8], Oc50A1.I.A [9] and CC1P1 [15] have further been shown to exhibit complement-fixing activities. The key functions of complement are the opsonization of the microbial surface promoting phagocytosis and activation of neutrophil chemotaxis. The importance of complement to host defence against the pneumococcus has been shown by the severe infections suffered by those with genetic complement deficiencies [33]. A clearance of S. pneumoniae requires both complement and the recruitment of neutrophils. Complement-mediated opsonization followed by ingestion and killing by professional phagocytes such as neutrophils or macrophages is a major host defence against S. pneumoniae [34]. BP-II, Oc50A1.I.A and CC1P1 might then, through their abilities to fixate complement and activate macrophages, be able to initiate an inflammatory process that facilitates the protection against pneumococcal infection. The previously reported protective effect of the pectic polysaccharide PM-II, isolated from the leaves of Plantago major L., against S. pneumoniae serotype 6B was also suggested to be due to pectic stimulation of the innate and not the adaptive immune system. No curative effect was observed when PM-II was given post-challenge [18].

Regarding the structure–activity relationship of immunomodulating pectins, it has been suggested that the β-D-(1 [RIGHTWARDS ARROW] 3,6)-galactan chains of AG-II play an important role in the immunomodulation of macrophages and that β-D-(1 [RIGHTWARDS ARROW] 4)-galactan chains of AG-I are important for the expression of effects towards both macrophages and Peyer's patch cells [10]. Arabinogalactan type II containing polysaccharides isolated from Artemisia tripartita and Juniperus scopolorum have shown to induce the production of IL-6 and MCP-1 from murine macrophages [35, 36]. A pectic polysaccharide, bupleuran 2IIc, isolated from the roots of Bupleurum falcatum L., has earlier been reported to enhance G-CSF secretion from intestinal epithelial cells. The active site for the expression of G-CSF enhancing activity was shown to be the RG-I region of the pectin [37].

BP-II has been reported to contain a pectic backbone comprising β-D-(1 [RIGHTWARDS ARROW] 4)-galacturonan and a rhamnogalacturonan core. The rhamnogalacturonan core seems to contain both AG type I and AG type II [10]. Oc50A1.I.A has been shown to contain heavily branched AG-II regions [9]. These structures in BP-II and Oc50A1.I.A might explain the previously reported immunomodulating activities and the protective effect against S. pneumoniae observed in the present study. CC1P1 did not elicit the same level of response as BP-II and Oc50A1.I.A, which might be explained by the simpler structure of this pectin, lacking both AG-I and AG-II type structures.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

In conclusion, our results suggest that i.p. administration of the pectic polysaccharide fractions BP-II, Oc50A1.I.A and CC1P1 protects against systemic S. pneumoniae infection in mice when given 3 h before challenge with the bacterium. This is demonstrated by means of lower levels of bacteraemia and lower scoring of adverse clinical signs in the animals treated with the pectic fractions, compared to PBS-treated mice. BP-II and Oc50A1.I.A, containing rhamnogalacturonan backbones with side chains of complex arabinogalactans, showed a stronger protective effect compared to CC1P1. CC1P1 consist of a simpler pectic structure, lacking both AG-I and AG-II type side chains.

The pectins showed no direct antibiotic effect towards S. pneumoniae, but stimulated pro-inflammatory cytokine production. Previously, BP-II and Oc50A1.I.A have been reported to stimulate macrophages and to fixate complement, and CC1P1 has been shown to exhibit complement fixation activity. The observed clinical effect is therefore thought to occur via the native immune system. The effect is probably independent of the putatively effect of the suspected LPS contaminant.

The use of the pectic fractions suggests that it is possible to increase the early inflammatory response and that this increase has a beneficial effect on survival. It is still not known whether the observed protection is due to increased neutrophil recruitment, enhanced macrophage and/or complement activation. The observed protective effects of the pectic polysaccharides are, however, promising in regard to the development of novel therapeutics to non-specifically augment innate immune responses. The pectins also do not cause significant side effects and have a low toxicity.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

This project has been financially supported by the Norwegian Research Council (Project no. 172292) and the EU 7th Framework Programme (‘Multi-disciplinary University Traditional Health Initiative (MUTHI)’ (Grant no. 266005)). Professor Eva Skovlund, School of Pharmacy, University of Oslo, is acknowledged for helping out with the statistical analysis.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information
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sji12047-sup-0001-FigS1-S8.docWord document2658K

Figure S1 Levels of IL-1β, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12 (p40) and IL-12 (p70) in mouse plasma from female NIH mice pretreated with BP-II or saline (PBS) (see Table 1).

Figure S2 Levels of IL-13, IL-17, TNF-α, IFN-γ, MIP-1α, MIP-1β, GM-CSF, RANTES and Eotaxin in mouse plasma from female NIH mice pretreated with BP-II or saline (PBS) (see Table 1).

Figure S3 Levels of IL-1β, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12 (p40) and IL-12 (p70) in mouse plasma from female NIH mice pretreated with Oc50A1.I.A or saline (PBS) (see Table 1).

Figure S4 Levels of IL-13, IL-17, TNF-α, IFN-γ, MIP-1α, MIP-1β, GM-CSF, RANTES and Eotaxin in mouse plasma from female NIH mice pretreated with Oc50A1.I.A or saline (PBS) (see Table 1).

Figure S5 Levels of IL-1β, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12 (p40) and IL-12 (p70) in mouse plasma from female NIH mice pretreated with CC1P1 or saline (PBS) (see Table 1).

Figure S6 Levels of IL-13, IL-17, TNF-α, IFN-γ, MIP-1α, MIP-1β, GM-CSF, RANTES and Eotaxin in mouse plasma from female NIH mice pretreated with CC1P1 or saline (PBS) (see Table 1).

Figure S7 Levels of IL-1β, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12 (p40) and IL-12 (p70) in mouse plasma from female NIH mice pretreated with LPS or saline (PBS) (see Table 1).

Figure S8 Levels of IL-13, IL-17, TNF-α, IFN-γ, MIP-1α, MIP-1β, GM-CSF, RANTES and Eotaxin in mouse plasma from female NIH mice pretreated with LPS or saline (PBS) (see Table 1).

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