Ethanolic Extract of Xestospongia Sp. Induces CD4+ and CD14 Cells Levels on Wistar Male Rat Infected with Staphylococcus aureus

Immunomodulator is a substance that increases or suppresses the immune response through the certain mechanism. The marine sponge Xestospongia sp. has immunomodulatory activity by increasing phagocytic activity. In addition, the phagocytic activity is affected by CD4+ and CD14 cells levels. Thus, this study aims to investigate the effect of Xestopongia sp. extract toward CD4+ and CD14 cells level in model rat. Animals were divided into 4 groups (n=5) and treated for 7 days, as follow: Group I (Ethanolic extract of Xestospongia sp. dose of 300 mg/KgBW); Group II (Ethanolic extract of Xestospongia sp. dose of 400 mg/KgBW); Group III (Phylantus niruri extract); and Group IV (0.5% Na CMC). On day 8, animals were intraperitoneally infected to Staphylococcus aureus and the blood was collected by cardiac puncture and assayed with ELISA kit CD4 (elabscience®) and ELISA kit CD14 (elabscience®). Ethanolic extract of Xestospongia sp. provided high levels of both CD4+ and CD14 cells (group II) compared to baseline (group IV) (p<0.05). Group I provided similar activity to group III (p>0.05) and group II provided significant activity with higher levels of CD4+ and CD14 cells compared to group III (p<0.05). In conclusion, both doses of Xestospongia sp. extract showed immunomodulatory activity by increasing CD4+ and CD14 cells levels, yet dose of 400 mg/KgBw provides the higher immunomodulatory activity.


Introduction
Immune systems act by body survival and protection against antigen that arise due to various materials from the environment. 1 Body's immune systems are able to identify and eliminate microorganisms that potentially harm to the body to prevent infections causing organs damage. 2 WHO (World Health Organization) stated that immune system disorder is the biggest problem in the worldwide. Immune system disorders are divided into primary and secondary disorders that caused by environmental factors, drugs and radiation 3 resulted in high sensitivity to infection, thus, immunomodulators can be used to increase the immune system as an alternative to control against infection.
Immunomodulator is a substance that changes activity of immune system, either by increasing or suppressing the immune response through certain mechanism. Their works in two ways, the stimulatory effect (increasing the immune response) or vice versa, the effect of suppression effect (suppress the immune response). 1,2,4 The use of terrestrial products such as marine sponge can be utilized in discovering novel drug such as immunomodulator agents. Marine sponges exhibit abundant biopharmacological activity such as antibacterial, antiviral, antifungal, antimalarial, antitumor, immunomodulator, and cardiovascular activity. 5 Xestospongia sp. reported has activity as immunomodulator by stimulating phagocytosis examined by its specific phagocytic activity. 6 Immune system involves phagocytic cells such as macrophages that play important role in the body's immune system against pathogens. One of the main roles of macrophages is phagocytosis, which aims to eliminate antigens, damaged or dead cells, and pathogenic bacteria such as CD 4+ and CD 14 cells. CD 4+ cells are antigen expres cells in the subset of thymocytes and T cell inflammatory cells (about 2/3 peripheral T cells), monocytes, and macrophages. 7,8 CD 4+ cells are type of white blood cells or lymphocytes which are important as part of the immune system. CD 4 or CD 4+ cells are referred to as T helper or T4 cells that activate Th1 cells thus activate macrophages in eliminating pathogens. 9 In other hand, CD 14 is Pattern Recognition Receptors (PRR) which enhance nonspecific immune responses to infections by increasing the sensitivity of immune cells to components of bacteria including lipopolysaccharide (LPS), lipoprotein and lipoteichoic acid (LTA). CD 14 delivers the bacterial component to various TLR (Toll-Like Receptors) on the surface membrane and induces immune cell activation. 11 Binding bacteria by the CD 14 receptor activate phagocytosis. 12 Thus, this study aims to examine the activity of ethanolic extract of Xestospongia sp. to phagocytosis activity observed from the increase in CD 4+ and CD 14 levels of rats models-treated with extracts and induced with Staphylococcus aureus. On day eight, each animal was infected intraperitoneally to 0.5 mL of Staphylococcus aureus, and left for 1 hour. After that, 3 mL of blood was collected intra cardial and put in EDTA-tube. Collected blood was centrifugated 3000 rpm for 15 minutes. Blood assayed with ELISA kit CD 4 (elabscience®) and ELISA kit CD 14 (elabscience®).

Data Analysis
Data was statistical analysis by using SPSS with ANOVA (Analysis of Variance) one-way test. p<0.05 value considered as significant in increased levels of CD 4 and CD 14 .

Results and Discussion
Immunomodulator is a substance that enhance immune response with various mechanisms. Both CD 4 and CD 14  According to Figure 1, all groups was significant to group IV as normal control cell). 6 The high levels of CD 4+ cell as well CD 14  According with results conducted at Figure  2, group II with 45.85±19.69 ng/mL provided high increasing levels of CD 14 compared to group I with 18.03±1.13 ng/mL, although both group was significant to group IV with 9.93±0.74 ng/mL as normal control (p<0.05). Group II was significant to group III as positive control (p<0.05) with 20.76±1.80 ng/ mL, meanwhile the group I was not significant with group III (p>0.05).
Previous study showed that ethanolic extract of Xestospongia sp. has activity as immunomodulator by increasing phagocytic activity of macrophages. Flavonoids contained in extract are suspected responsible in increasing phagocyctic activity by activating Th1 cell assisted with T cell helper (CD 4+