Veterinary World, EISSN: 2231-0916 RESEARCH ARTICLE Available at www.veterinaryworld.org/Vol.12/June-2019/1.pdf Open Access Immunomodulatory potential of polysaccharides from Coriolus versicolor against intracellular bacteria Neisseria gonorrhoeae Manikya Pramudya and Sri Puji Astuti Wahyuningsih Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia. Corresponding author: Sri Puji Astuti Wahyuningsih, e-mail: [email protected] Co-author: MP: [email protected] Received: 14-12-2018, Accepted: 09-04-2019, Published online: 01-06-2019 doi: 10.14202/vetworld.2019.735-739 How to cite this article: Pramudya M, Wahyuningsih SPA (2019) Immunomodulatory potential of polysaccharides from Coriolus versicolor against intracellular bacteria Neisseria gonorrhoeae, Veterinary World, 12(6): 735-739. Abstract Background and Aim: For many years, people use natural products from the plant and fungal to improve immune response against microorganism. This study aimed to investigate the immunomodulatory properties of polysaccharides (PS) from Coriolus versicolor in mice infected by intracellular bacteria Neisseria gonorrhoeae. Materials and Methods: Thirty-six female BALB/C mice were divided into six groups: Normal control, negative control, positive control, P1 (PS before infection), P2 (PS after infection), and P3 (PS before and after infection). PS were administrated for 10 days. N. gonorrhoeae was infected twice with 2 weeks gap from the first to second exposure with a dose of 106 cells. 1 week after the end of treatment, level of oxidants, innate immune responses, and adaptive immune responses were measured. Results: This study showed that PS administration could restore the number of leukocytes as normal but could not enhance the number of phagocytes and its activity. PS administration also showed immunosuppression activity by lowering nitric oxide levels in P2 and P3 groups (p<0.05). This result showed that PS prevent over-expression of pro-inflammatory cytokines by decreasing phagocytic activity. Contrast with innate immune response result; PS administration could significantly increase interferon-gamma level in P1, P2, and P3 groups (p<0.05). Level of antibodies was significantly increased in the P3 group (p<0.05). PS administration also showed an increased level of tumor necrosis factor-α, but the difference was not significant (p>0.05). Conclusion: PS enhance adaptive immunity due to the capability of N. gonorrhoeae that able to survive and replicate in phagocytes. Thus, PS from C. versicolor could be potentially be used as a natural immunomodulator against intracellular bacteria. Keywords: immune response, immunomodulator, Neisseria gonorrhoeae, polysaccharide. Introduction N. gonorrhoeae, but these bacteria are relatively resis- Sexually transmitted infection such as gonor- tant to degradation of phagocytes and able to modu- rhea is one of the major public health problems which late apoptosis in macrophage [7,8]. Immune response has been causing serious morbidity and mortality [1]. mediated by T-cell is needed to destroy these bacte- Neisseria gonorrhoeae infections are approximately 60 ria. The human body needs a particular compound to million cases each year worldwide [2]. Hananta et al. [3] modulate immune response. reported that the prevalence of asymptomatic urogen- Polysaccharides (PS) from natural sources such ital gonorrhea among the Indonesian population is as fungal and plant have been known to improve the very high. N. gonorrhoeae infects the female cervix immune function of the body [9,10]. Coriolus versicolor and male urethra leads to severe complications such is one of the medicinal mushrooms used in Japan, China, as pelvic inflammatory disease, urethritis, cervicitis, Korea, and other Asian countries [11]. Both PS krestin ectopic pregnancy, disseminated gonococcal infection, and PS peptide from C. versicolor are active as biological and infertility [4]. Moreover, N. gonorrhoeae infec- response modifier. Carbohydrate in powdered polysac- tion is associated with increased risk of HIV trans- charide krestin contains 91-93% active compound β-glu- mission [5]. N. gonorrhoeae are intracellular bacteria can [11]. Wahyuningsih et al. [12] reported that PS of that able to survive and replicate inside of the cell [6]. C. coriolus could enhance the response of immunoglob- Naturally, innate immunity can inhibit transmission of ulin against Pseudomonas aeruginosa. Another study was also reported that polysaccharide from C. versicolor Copyright: Pramudya and Wahyuningsih. Open Access. This induce B-cell activation and enhance cytokine produc- article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons. tion [13]. However, studies have not been reported on the org/licenses/by/4.0/), which permits unrestricted use, distribution, activity of PS from C. coriolus as an immunomodulator and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to against N. gonorrhoeae in Indonesia. the Creative Commons license, and indicate if changes were made. Therefore, this study aimed to investigate The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data the immunomodulatory properties of PS from made available in this article, unless otherwise stated. C. versicolor growth in Indonesia including number Veterinary World, EISSN: 2231-0916 735 Available at www.veterinaryworld.org/Vol.12/June-2019/1.pdf of phagocytes, number of leukocytes, phagocytic Phagocytes and leukocytes counts activity, level of cytokine, level of antibody, and level Blood sample (10 µL) and intraperitoneal fluid of nitric oxide (NO) in mice infected by intracellular (30 µL) were dissolved with 100 µL of Turk solu- bacteria N. gonorrhoeae. tion, separately. Then, number phagocytes and leuko- cytes from both blood and intraperitoneal fluid were Materials and Methods counted using hemocytometer. Ethical approval Phagocytes activity assay All procedures involving animal care were car- The intraperitoneal suspension (70 µL) was ried out in accordance with the guidelines laid down smeared on glass slides and air-dried. The smear was by Animal Care and Use Committee of Veterinary fixed using methanol for 15 min and stained with Faculty, Universitas Airlangga, Surabaya, Indonesia. Giemsa solution for 20 min. Phagocytic activity was Materials and chemicals determined by counting the number of phagocytes in C. versicolor was collected from Surabaya, Kediri a population of 100 phagocytes. and Tulungagung, East Java, Indonesia. N. gonor- Serum cytokines and antibody assay rhoeae was purchased from Balai Besar Laboratorium Whole blood was collected and centrifuged at Kesehatan, Surabaya Indonesia. Antibody level, inter- 3000 rpm and 4°C for 10 min, while the upper layer feron-γ (IFN-γ), tumor necrosis factor (TNF)-α, and contained the serum. The levels of antibody, IFN-γ, and enzyme-linked immunosorbent assay (ELISA) kit TNF-α, in the serum, were analyzed by commercial were purchased from BioLegend (BioLegend, Inc., ELISA kits (BioLegend, Massachusetts, USA) accord- San Diego, USA). All other chemicals and solvent ing to the manufacturer’s protocol. Level of cytokines used were of analytical reagent grade. IFN-γ and TNF-α was measured using Sandwich- Preparation of crude PS from C. versicolor ELISA method. Meanwhile, the level of antibody was According to Cui and Christi [11], C. versicolor measured using an indirect ELISA method. The absor- in coarse powder (200 g) was macerated twice with bance was measured using the ELISA reader at 450 nm. o 3 L and 1 L of water, heated at 80-98 C for 2-3 h. The NO assay sample was filtered using Whatman paper No.41, Deproteinated serum (50 µL) was added with vacuum and Buchner funnel and supernatant were 100 µL Griess reagent I and 100 µL Griess Reagent II. collected. The supernatants were precipitated by After that, the sample was homogenized using vortex o ammonium sulfate 90% for 24 h at 4 C. Then, the and incubated for 10 min at room temperature. The o sample was centrifuged at 9000 rpm for 30 min at 4 C. absorbance was measured at 540 nm. The precipitated material was then dissolved in phos- phate-buffered saline (PBS) (30 mL) and dialyzed Statistical analysis through nitrocellulose membrane for 24 h in PBS at Statistical analysis was performed by one-way 4oC. The aqueous solution was freeze-dried to obtain analysis of variance followed by Duncan’s post hoc polysaccharide from C. versicolor (PS). test. All analyses were performed using SPSS Statistic 24 Software (IBM Corporation, USA). The results Animals were reported as the mean±standard deviation of six Thirty-six female BALB/c mice (8-10 weeks; repeats. p<0.05 was considered statistically significant. 30-40 g) were obtained from Faculty of Pharmacy, Universitas Airlangga (Surabaya, Indonesia). The Results animals were maintained in cages made of plastic at Number of phagocytes 20oC, with 12-h light/12-h dark cycle, fed and watered The number of phagocytes was significantly by ad libitum. increased in KN–group compared to normal control (p<0.05). All of the treatment groups with the admin- Experimental design istration of C. versicolor PS showed no significant After 7 days of acclimatization, mice were ran- difference compared to control groups. P3 group domly divided into six groups (KN: Normal control; (114±23 cell/mm3) showed