Stimulation of Phagocytosis in Alveolar Macrophages and Resistance against Infection in Mice by the Aqueous Extract of Rhizomes of Aframomum danielli, Schum (zingiberaceae)

Oumar Mahamat^1*, Tume Christopher^2,3, Guessom Oulianovie⁴ and Kamanyi Albert^5
*Correspondence: Oumar Mahamat, Department of Biological Sciences, Faculty of Science, University of Bamenda, P.O. Box 39 Bambili, Cameroon, Tel: +002376765697-18, Email:

Keywords

Aframomum danielli; Alveolar macrophages; Phagocytosis; Immunostimulation

Introduction

In developing nations, where, malnutrition and infectious diseases remain a challenge, a stimulation of body defense mechanisms is of high interest. Malnutrition and infectious diseases contribute to cause immunodeficiencies.

Immunomodulating drugs are therefore necessary to control various opportunistic infections such Staphylococcus aureus infection [1-4]. In respiratory tract, alveolar macrophages are frequently target of immunomodulating drugs to boost the resistance against infections [5,6].

Medicinal plants such as Pterocarpus erinaceus, Barleria prionitis, Pseudocedrela kotschyi, Ficus septica have been claimed to possess immunomodulatory activities [7-10]. Plant such as Aframomum danielli is cited in control of diseases related to immune disorders, however it is not been studied on alveolar macrophages despite its common use in management of respiratory tract infections [11-14].

As, alveolar macrophages, constitute the first line of defence against microorganisms carried out by alveolar macrophages, the present investigation is designed to assess the effect of aqueous extract of A. danielli rhizomes on alveolar macrophages functions through evaluation of phagocytosis and killing of Staphylococcus aureus in mice [15].

Materials and Methods

Study design and setting

The study protocol was approved by the Ethics Committee for animal use in research studies of the laboratory of the Department of Biological Sciences, University of Bamenda, Cameroon.

The study was conducted at the Institute of Agricultural Research for Development (IRAD) of Bambui, Cameroon. All procedures involving animal were in accordance with the Principles of Laboratory Animal Care [16].

Experiment animals

Male albinos white mice (Mus musculus) originated from Laboratoire National Veterinaire (LANAVET) were used for isolation of macrophages and in vivo tests. They were housed in plastic cages with softwood shavings and chips as beddings, with free access to pellet diet and clean drinking water. Animals were acclimatized to the working environment 1 week before the beginning of the experiment. At end experiment, they were anesthetized by a mixture of ketamine chloride (80 mg/kg) and xylazine chloride (3.2 mg/kg) injected intraperitoneally.

Microbe strains and infection

Clinical isolates of C. albicans and S. aureus used were got from the laboratory of Biochemistry, University of Dschang, Cameroon. They were administered to mice intravenously via the tail or intranasal (1 × 10⁸ microorganisms/animal).

Plant collection and extraction

Aframomum danielli specimen was collected from Bambili, Cameroon. The plant was identified by a taxonomist at the Department of Biological Sciences and confirmed by the National herbarium Yaounde, Cameroon. Plant rhizomes were collected, chopped into small pieces, washed with clean water and air dried at 38°C. They were powdered and boiled in distilled water for 1 hour. After filtration, using Whatman paper No 1, the filtrate was evaporated at 35°C in the ovum and the resulting dried product was collected and used after dissolution in PBS.

Isolation of Alveolar Macrophages

Alveolar macrophages (AMs) were obtained by lavage of lungs from five mice. Prior to the date of isolation (20 h), mice were given a single S.C. dose of saline of dexamethasone disodium phosphate (Dex, Sigma Chemical) of 20 mg/kg. The lungs were excised and washed 5 times with RMPI-1640 using brief massage. Collected cells were estimated to about 5-10 × 10⁶ cells (80% macrophages) for the 5 mL lavage fluid. After centrifugation (10 min, 1800 tr/min) at 37°C, cell pellet was suspended in complete RMPI-1640 medium (medium supplemented with 5% bovine serum fetal, 100 units/mL penicillin and 100 μg/mL streptomycin). The number of cells was counted using a malassez hemocytometer. The viability of the cells (≥ 98%) was determined by trypan blue (Difco) exclusion assay.

Slide methods for evaluating the phagocytosis

Human blood (0.4 mL) was obtained by finger prick method on a sterile glass slide and incubated at 37°C for 25 min to allow clotting. Later, the clot was carefully removed, and the slide was slowly drained with sterile PBS, taking care not to wash the adhered phagocytes. Next, the slide was flooded with 1 mL of extract and incubated at 37°C for 20 minutes. Slides were once more flooded with 1 mL of suspension of opsonized C. albicans prepared in normal saline solution mixed with human serum (ratio 8:2) and incubated at 37°C for 30 minutes. Finally, the slide was drained fixed methanol and stained with giemsa stain (the procedures were repeated and the slide stained after 1 hour).

The mean number of phagocytosed cells on the slide was determined microscopically for 100 phagocytes using morphological criteria. This number was taken as the Phagocytic Index (PI) and was compared with basal PI of controls. Plant effect expressed in percentage was calculated using the following equation:

Stimulation (%) = PI _(test) - PI _(control) × 100/PI _(control) [17]

Bacteria killing assay

The killing assay for S. aureus was adapted from a method of measurement of intracellular killing of bacteria [18]. AMs were cultured in T75 flasks (NUNC) for 48 hours in RMPI-1640 with the extract or levamisole (4 μg/mL) as control positive. Later, they were cultured in triplicate in a 96-wells microplate with a blank well containing the medium (2 × 10⁷ macrophages/ mL). To each well, it was added 100 μL bacterial suspension in RPMI contenaing 3% Normal Human Serum (NHS) and incubated at 37°C under gentle shaking for 90 min. Bacteria was added in ratio macrophages/bacteria of 1:4 and the final volume of the mixture was 150 μL per well. From each well, 100 μL of the culture was collected and transferred to other microplate, immediately after adding the bacterial suspension (T₀) and 60 min after (T60), and placed on ice. At 90 min (T₉₀), the initial microplate was also placed on ice.

Serial 10-fold dilutions were made in ice-cold H₂O of the three cultures and plated on LB agar plates. After overnight culture at 37°C the numbers of colonies were counted. The killing was reflected by a decrease in number of colonies at T₆₀ and T₉₀ relative to the number of colonies at T₀. The killing percent was calculated using the following formula:

% Killing = 100 × {1-(Number of colonies at T₆₀ or T₉₀)/(Number of colonies at T₀)} [19]

Cell culture and ROS assay

The purified alveolar macrophages (2 × 10⁵ cells/well in 96 well tissue culture plate) of Dex-treated mice were incubated with RMPI 1640 containing 1 μg/mL lipopolysaccharide (LPS) and extract at different concentrations for 24 hours at 37°C. Macrophages from Dex-untreated mice were used as positive control. Nitric oxide (NO) and hydrogen peroxide (H₂O₂) were determined in the macrophage culture supernatants, while superoxide anion (O₂^-) was measured in cells by colorimetric methods. The activity of the extract was determined and expressed as percentage of stimulation.

Measurement of AM superoxide anion synthesis

The assay used was the ability of the superoxide produced by macrophages to reduce yellow nitroblue tetrazolium (NBT) to blue formazan [20]. To the cells, 50 μL of NBT solution (1 mg.ml^-1) was added and incubated for 1 h. Then, washed 3 times with PBS 1X and cells were fixed with methanol 100% for 3 min. After fixation, cells were washed twice with 70% methanol and allowed to air dry. The formazan produced was solubilized by adding 120 μL of 2M KOH and 140 μL 100% DMSO. Absorbance was read at 630 nm using KOH/DMSO as blank. The optical densities were used as follows to determine the effect of the extract:

% activity = 100 × {(OD _sample - OD _control)/OD _control}

Measurement of AM NO oxide production

The nitrite/nitrate content, indicative of NO production by iNOS, was monitored by the Greiss reagent assay [9]. In 96-well plates, supernatant samples ( 100 μL) were mixed with 100 μl Greiss reagent, consisting of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-napthyl) ethylenediamine dihydrochloride (1:1 vol/vol) and incubated for 10 min. The change in absorbance was monitored at 545 nm and the optical densities were used to determine the % activity.

% activity = 100 × {(OD_sample - OD_control)/OD_control}

Measurement of AM hydrogen superoxide release

H₂O₂ was analyzed using the colorimetric method [21]. To 50 μL 10 mM FeSO₄•7H₂O (dissolved in 1 mM HCl) and 25 μL 2.5 mM KSCN (dissolved in PBS) were added to 100 μL of the supernatant. Optical densities were measured in a microplate reader at 490 nm wavelength. Additionally calibration curve were assessed. Optical densities of RPMI 1640 containing various H₂O₂-concentrations (0-200 μM f.c.) and 20 μL TCA (50%) were measured and used for standard curve for determination of the H₂O₂ release in tested well. The % activity was determined as follows:

% activity = 100 × {(H₂O₂)_sample - (H₂O₂)_control)/(H₂O₂)_control}

Evaluation of the effect of extract on death rate induced by S. aureus infections in dexamethasone-treated mice.

Mice (Mus musculus) were treated with dexamethasone 20 mg.kg^-1, 20 hours prior to infection. Later, they were infected with lethal inocula 1 × 10⁸ S. aureus and daily treated P.O. with extract for 5 days. Levamisole (25 mg/kg) was administered to the control. Mice were observed for 28 days. From day 6 post treatment, the survival percentage and survival time for each mouse was recorded. The percentage of survival was calculated as follows:

% Survival = 100 × (Number of survival/Total number of group)

Antibacterial activity tests of extract in repository, suppressive and curative treatments against S. aureus in mice.

Swiss albino mice (18-25 g) were randomly divided into different groups of 5 mice per cage for the various treatments as described in Table 1. Extract, levamisole and distilled water were orally and daily administered by gavage for 7 days. Animal were intra-nasally inoculated and S. aureus (1 × 10⁸ microorganisms in PBS) was used as microorganisms. Nasal lavages were obtained as described by Bogaert et al. and the lavage product was inoculated on LB agar [22]. The bacteremia level was assessed by counting the number of colonies 24 hours of incubation at 37°C.

Statistical analysis

All statistical analyses were done on GraphPad Prism version 5.02. Data were expressed as mean ± Standard Deviation (SD). ANOVA test was used to analyze the difference among various treatments with Least Significance Difference (LSD) at 0.05 followed by student Newman-keuls’s post-test.

Results

Stimulation of C. albicans ingestion by human peripheral blood phagocytes

Results as presented in Table 2 showed that the extract, concentrationdependently and time-independently, increased the phagocytosis of opsonized C. albicans (p<0.05). Used of heat-inactivated serum for opsonisation did not change the extract-mediated enhancement of phagocytosis (data not shown), indicating complement independency.

Stimulation of killing of S. aureus by Alveolar macrophages

Aqueous extract of A. dalnielli increased significantly (p<0.05) the intracellular bacteria killing activity of Dex-treated macrophages compared to untreated macrophages (Table 3).

The % killing of extract-treated macrophages was concentration dependent at any time point with comparable percent to those of macrophages pre-incubated with levamisole at 160 μg/mL and 320 μg/mL.

Effect of A. dalnielli on production of reactive oxygen species

Data in the present study (Table 4) showed that extract of A. danielli elicited a dose-dependent significant (P<0.05) increase of the production of superoxide anion production, nitric oxide and hydrogen peroxide by macrophages of Dex-treated mice compared to untreated macrophages.

Protection of dexamethasone-treated mice against S. aureus

Treatment with aqueous extract of A. dalnielli showed significant resistant of mice against S. aureus (Figure 1). At 21 days, all saline water treated mice dead while 40%, 60% and 90% survived and were cured in groups treated with 100, 200 and 400 mg/kg extract (P<0.05).

Treatments of experimentally induced S. aureus infection in mice

Aqueous extract of A. danielli at 100, 200 and 400 mg/kg, in repository treatment as well as in suppressive and curative treatment, caused a dosedependently suppression of bacteremia against S. aureus reducing the density of bacteremia compared to control (Figure 2). Similarly, levamisole caused significant suppression of bacteremia density in repository, suppressive and curative treatment.

Discussion

In a therapeutic dose 0.05 mg/kg, dexamethasone results in reduction of AMs as well as depression of their phagocytic activity [5]. It inhibits the functions of blood leucocytes and tissue macrophages by modulating the release of cytokines, inflammatory mediators and ROS generation, avoiding that the animal’s own immunologic response to the disease cause intense tissue damage and further aggravation of the condition [15,23]. In the present study, our analysis provided clear evidence that the extract of A. dalnielli increases level of microorganism’s ingestion by human blood cells as well as stimulates the killing of S. aureus by alveolar macrophages of Dex-treated mice. This demonstrates that the extract of A. dalnielli the extract stimulates the activities the phagocytic activities AMs.

Dexamethasone, in the 0.05 mg/kg therapeutic dose, was described to reduce the oxidative metabolism of healthy calves’ alveolar macrophages [15]. Here, we found that AMs of Dex-treated mice produced more of superoxide anion, hydrogen superoxide and nitric oxide when treated with the extract A. dalnielli rhizomes increased the production. This might justify the effectiveness of the extract shown in bacterial killing as reported by numerous studies [24].

Reactive oxygen species are generated in macrophages mainly by following mechanisms: xanthine oxidase-dependent, respiratory chain and NADPH oxidase-(NOX-) dependent pathways [25]. Extract of A. dalnielli has increased ROS production suggesting that it might be acted by activating one of these pathways. Macrophages NO production was significantly increased by extract of A. dalnielli. As NO produced by iNOS in macrophages and neutrophils serves an important pathological and immunological role, this suggest that extract A. dalnielli rhizomes might activate iNOS in macrophages as is inhibited by dexamethasone [26].

In vivo test, he extracts significantly increased survival percent of Dextreated mice infected with S. aureus, reducing the bacteraemia level. This suggests that the extract of A. dalnielli rhizomes might act not only on alveolar macrophages but it could affect peripheral blood polymorphonuclear or mononuclear phagocytes. In curative, suppressive and prophylactic treatment against nasal infections by S. aureus in Dex-treated mice, the extract of A. dalnielli significantly exhibited suppressive, prophylactic and curative activities confirming that extract stimulate the phagocytosis in alveolar macrophages.

Conclusion

The findings of this study supported by the traditional uses, sustain that A. danielli rhizomes are important in control of pulmonary infections via its stimulatory effect on macrophages, when S. aureus is known as one of the most common causes of bacterial lung infections in normal hosts.

Acknowledgement

The authors wish to acknowledge Irad Bambui, Cameroon, for the assistance received by offering the laboratory where part of the research was done. The authors want to thank particularly Miss. Hakoueu Flora for disposal when it was needed.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Availability of Data and Materials

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Authors Contributions

OM was participated for conception of ideas, experiments, data analysis and interpretation, and preparing the manuscript. GKO was also participated for conducting the experiments. TC was participated for conception of ideas. KA was participated for conception of ideas. All authors read and approved the final manuscript.

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