THE EFFECT OF STEEPING ROBUSTA COFFEE BEANS ON MONOCYTES: EXPRESSION OF IL-1β AND TNF-α AGAINST Streptococcus mutans

Adhesion, IL–1β, TNF–α are components that affect in inflammation. So, the effect of steeping green and black Robusta coffee beans to adhesion of Streptococcus mutans on this components. This study used monocytes isolated from healthy human peripheral blood using Ficoll-Hypaque centrifugation method. Monocytes were divided into eight groups, i. e. (i) Control group (untreated monocytes), (ii) S. mutans group (monocytes + S. mutans), (iii) Black Coffee 2.5 % group (monocytes + black coffee beans 2.5 % + S. mutans), (iv) Black Coffee 5 % group (monocytes + black coffee beans 5 % + S. mutans), (v) black Coffee 10 % group (monocytes + black coffee beans 10 % + S. mutans), (vi) Green Coffee 2.5 % group (monocytes + green coffee beans 2.5 % + S. mutans), (vii) Green Coffee 5 % group (monocytes + green coffee beans 5 % + S. mutans), (viii) Green coffee 10 % group (monocytes + green coffee beans 10 % + S. mutans). S. mutans adhesion on monocytes was analyzed using histochemistry method, while immunocytochemical staining was used for analyzing IL–1β and TNF–α. Cells counting was done per 100 monocytes under a light microscope with 400 x magnification. Data were analyzed using ANOVA followed by LSD test. Results showed that steeping green and black Robusta coffee beans increased the adhesion of S. mutans on monocytes, but it decreased of IL–1β, TNF–α expression (P <0.05). In conclusion, steeping of Robusta coffee beans increased adhesion and decreased IL-1β, TNF-α against S. mutans.

Robusta coffee beans have the function of anti-inflamatory activity based on previous studies. It increased the cells viability, inhibited to growth S. mutans, decreased inflammatory cell count (in vivo). Robusta coffee also increased the number of fibroblast cells and decreased the expression of IL-1α in vitro and in vivo (DEWANTI, 2016).
Among those inflammatory activities is phagocytosis. It is one of the immune system against pathogens such as S. mutans. The process of 1.2 Department of Biomedical Science -Faculty of Dentistry of Jember University -Jl. Kalimantan No. 37 Jember 66131 -East Java Indonesia -idewadewanti@yahoo.com, el_pujiana.fkg@unej.ac.id 3.4 phagocytosis as follows: (i) the recognition, which is a process in which foreign microorganisms or particles are detected by phagocyte cells. (ii) the movement (chemotaxis), phagocyte cells move toward the pathogen. (iii) the adhesion, pathogen will attached to the receptors on the phagocyte cell membrane. (iv) the ingestion, the process of ingesting pathogens into the cytoplasm, which will enter the cytoplasm inside a vacuole-like bubble called the phagosome. (v) the digestion, lysosomes containing destructive enzymes such as acid hydrolase and peroxidase, will fuse with phagosomes to form phagolysosomes, then digest foreign matter. Sixth, the secreting, the remaining product of foreign particles that are not digested will be excreted by phagocyte cells (ABBAS et al., 2015).
Regarding the process of Phagocytosis is inflammation. Accordingly, important chemical mediator in inflammation is TNF-α and Il-1β. TNF-α ascachectin is a strong proinflammatory cytokine and plays role in the immune system. Inflammation must occur, but it also causes damage to cells because it can release of chemical mediators, phagocytic enzymes (phagocyte The effect of steeping robusta coffee beans on ... 478 (monocytes + green coffee beans 2.5 % + S. mutans), (vii) Green coffee 5 % group (monocytes + Green coffee beans 5 % + S.mutans), (viii) Green Coffee 10 % group (monocytes + green coffee beans 10 % + S. mutans). All groups were incubated for 24 h at room temperature. Monocytes were made preparates and fixation with methanol. S. mutans adhesion on monocytes was analyzed using histochemistry method (Giemza staining), while immunocytochemical staining was used for analyzing IL-1β and TNF-α. Immunocytochemical analysis were carried out in the following ways: the preparation was soaked in blocking solution with peroxidase at room temperature for 10 min, then incubated in back-ground sniper (protein blocking solution) for 10 min at room temperature. Primary antibodies were added 2 × 10 -5 L, incubated at 25 °C for 60 min and washed with PBS. Secondary antibodies were added, incubated and washed with PBS. Preparation added with Trek Avidin-HRP reagent, washed with PBS, preparation with DAB chromogen substrate, washed with tap water. Hematoxylin mayer (counterstain) was added to the preparation, then incubated for 1 min to 3 min, then washed under tap water and dried. Cells counting was done per 100 monocytes under a light microscope with 400× magnification. Data were analyzed using ANOVA followed by LSD test.

RESULTS AND DISCUSSION
Results of this study showed in figures and tables. Figure 1 showed S. mutans looked around the monocytes. Adhesion (Figure 1 and Figure 2). Analysis with ANOVA and LSD showed a difference (P < 0.05) ( Table 1). Whereas LSD analysis (P < 0.05) there was a significant difference between the control group and the black and green coffee group and between the S. mutans group and the black and green coffee groups. On the other hand, it was no significantly different between groups of the black coffee and the green coffee. The higher concentration of steeping black and green robusta coffee beans, the more number of S. mutans were attached on monocytes.
The result of IL-1β and TNF-α expression described as brown in cytoplasmic of monocytes, but also expressed on extracellularly, so next it must be analyzed to know level of these cytokines (Figures 3 and Figure 5). ANOVA (P < 0.05) showed that a significant difference between groups, while LSD (Table 2 and Table 3) showed that no significantly different between the black coffee and the green coffee (P < 0.05).
So the higher of the concentration of oxidase, inducible nitric oxide synthase, and lysosomal protease), free radical compounds and superoxide (BRADLEY, 2008;HANA & JAN, 2013;CHARLES, 2011). The Il-1β family has been extensively reviewed in various literature having many roles in acute and chronic inflammation. The cytokine interleukin-1β (IL-1β) like TNF-α is a plays role as a mediator of the inflammatory response that plays an important role in the host's response. However, it also causes damage during chronic diseases and acute tissue injury (CHARLES, 2011;GLORIA & DAVID, 2011). The aim in this study is to analyze the effect of black and green of steeping robusta coffee beans on adhesion of S. mutans on monocytes and the expression of IL-1β, TNF-α in monocytes. The following is the methods of the research. Peripheral blood from healthy people as much as 6 × 10 -3 L was mixed with anticoagulant (heparin). Then, the blood was layered on Ficollhypaque and centrifugated (198.968 rad s -1 , 30 min, 26 °C). The monocyte layer was then taken and add with HBSS in the ratio of 1:1. The next step after pipetting, it was centrifugated (178.024 rad s -1 , 10 min, 26 °C). The supernatant was discarded and add with HBSS, Fungizone 5 × 10 -6 L, and Penstripe 2 × 10 -5 L, then it was incubated for 24 h at room temperature. Monocytes were then layered inside the culture dish and added with RPMI. Afterward, cells were placed on 24well microtiter plate 8 × 10 5 cells/well, then it incubated for 45 min 37 °C, then it was washed 4× with HBSS medium. Monocytes were divided into eight groups, i. e. (i) Control group (untreated monocytes), (ii) S. mutans group (monocytes + S. mutans), (iii) Black Coffee 2.5 % group (monocytes + black coffee beans 2.5 % + S. mutans), (iv) Black Coffee 5 % group (monocytes + black coffee beans 5 % + S.mutans), (v) black Coffee 10 % group (monocytes + black coffee beans 10 % + S. mutans), (vi) Green Coffee 2.5 % group Dewanti, I. D. A. R. et al. 479

MATERIAL AND METHODS
FIGURE 1 -Adhesion activities S. mutans in monocytes after exposed by steeping of green and black and green robusta coffee beans. Analyzed with a light microscope with magnification 1 000 x. Adhesion activities were shown with S. mutans that surround a monocyte cells (black arrow). Monocytes were lysis (red arrow).      FIGURE 6 -Diagram of TNF-α of monocytes after exposed by steeping of green and black Robusta coffee beans and S. mutans.

Sum of
Coffee Science, Lavras, v. 14, n. 4, p. 477 -483 oct./dec. 2019 The effect of steeping robusta coffee beans on ... 482 steeping green and black Robusta coffee beans caused decreasing of IL-1β, TNF-α ( Figure 2 and Figure 4). Steeping green and black Robusta coffee beans caused decreasing of inflammation against S. mutans. The result showed that S. mutans group appears fewer adhesion activities than the coffee group and many monocytes were lysis, it suspected monocytes cells were not against to S. mutans with a maximum. Cell damage can be caused by cell-derived NO resulting in cellular respiration disorders, cell function and proliferation (cytostatic). NO will bind to Ferrum and prevent Ferrum from leaving the cell causing host cell damage (cytocidal) (ALLAIN et al., 2011). The coffee groups were proved that the higher the concentration, the higher the adhesion activity of S. mutans in the monocytes cell. Besides that, very few monocytes cells undergo lysis. That process was thought to be suspected by the bioactive components of coffee beans that also have antibacterial, and it could maintain cells survival. Robusta coffee beans could increase cells viability (DEWANTI, 2003). Antioxidants can inhibit the action of cytokine-induced NO synthase (iNOS) enzymes through iNOS control of mRNA and inhibit the transport of arginine by the control mechanism of CAT-2 mRNA (cationic amino acid transporter-2 mRNA) (FRANCESCHELLI et al., 2019).
Bioactive components of coffee beans were flavonoids, cafein, chlorogenic acid, and alkaloids (RAMANAVICIENE et al., 2003). These components were alleged had role as an immunomodulator. In studies of other natural ingredients that contain flavonoids have the ability to improve the immune system. A study of in vivo cellular immunity function in mice proves that flavonoid compounds can stimulate lymphocyte proliferation, increase T-cell count and increase IL-2 activity. Flavonoids potentially work against lymphokines produced by T cells that will stimulate phagocyte cells including monocytes to perform phagocytic responses (SHEN & JU-HUA, 2018). Monocytes have receptors that can recognize S. mutans. The major receptors known to play a role against S, mutans is Dectin-1, TLR2, and TLR4. Dectin-1 induces phagocytosis whereas TLR2 induces activation of cytokine production (NETEA et al., 2006;DENNEHY et al., 2009). Coffee beans are thought to bind to receptors on monocyte cells that affect the transcription proteins and cell nuclei, subsequently increasing activity of Dectin-1, TLR2, and TLR4 receptors of monocytes cells thus increasing activity in recognizing S. mutans, thus increasing the number of monocytes cells active. Also, monocytes cells release cytokines such as IFNγ, IL-1β, and TNF-α which are known to be factors that trigger adhesions, especially IL-1β known as immunoregulators can stimulate the expression of intercellular adhesion molecule-1 (ICAM-1), ICAM-1 causes monocyte to easily adhesion (THICHANPIANG et al., 2014).

CONCLUSION
Steeping of Robusta coffee beans increased adhesion and decreased IL-1β, TNF-α against S. mutans. If the concentration is higher, so the adhesion activities is higher. So, steeping of green and black Robusta coffee beans reduce inflammation caused by S. mutans.