Aqueous Leaves Extract of Gongronema latifolium (Benth) Downregulates the Expression of IFN-γ , IL-10 and Cell Surface Markers in Rabbits

Background: The pathophysiology of the inflammatory process reveals intricate signaling which includes the IL-1β, IL-6, and TNFα pathways that could serve as drug targets. Aim: This study determined the effect of the aqueous extract of Gongronema latifolium (AEGL) leaves on the expression of IFNγ, IL-10, CD3, and CD56 in rabbits. Materials and Methods: ELISA tests were performed to determine the effect of the AEGL on the expression of a pro-inflammatory cytokine (IFNγ), an anti-inflammatory cytokine (IL-10), and CD3 and CD56 cell surface markers in rabbits. Twenty cross-bred male rabbits


Introduction
Inflammation is an intricate biological process that involves the protection of the body from invading factors and at times marks the onset and/or the progression of diseases [1]. Inflammation involves a vast array of cells, cytokines, chemokines, acute phase proteins, cell surface markers, and immune mediators [2]. The inhibition of any specific protein in these cascades could serves as a viable drug target in the alleviation of inflammatory conditions. To maintain homeostasis, anti-inflammation is facilitated by certain cytokines and mediators and intricate signaling pathways [2]. In order to promote anti-inflammation, these immune agents must be activated. Food, herbs, drugs, and biologics offer a vast array of small molecules, peptides and proteins that have either pro-or anti-inflammatory activity [3].
The T Lymphocytes (T cells) and Natural Killer (NK) cells with CD3 and CD56 markers respectively are important players in the adaptive immune system. Through the production of cytokines and other immune mediators, they play crucial role in the process of inflammation and immunomodulation [4] [5]. IFN-γ and IL-10 are key cytokines that alter the immune system with their proinflammatory and anti-inflammatory activities respectively [6]. The IL-10 or human cytokine synthesis inhibitory factor (CSIF) is a multifunctional cytokine that regulates immune and inflammatory responses. It is an anti-inflammatory cytokine which is released during systemic infections. It is produced by NKC cells, macrophages, dendritic cells (DC), B cells, and CD4+ T cells (Th2) [7]. IL-10 exerts pleiotropic effects on hemopoietic and non-hemopoietic cells such as by endothelial cells and keratinocytes [7]. It affects immunosuppression by the inhibition of the effector functions of NKC cells, macrophages, monocytes and Th1 cells. It does this by inhibiting the maturation and differentiation of these haemopoeitic cells and subsequently their cytokines and chemokines [7] [8]. IL10 binds to and forms a complex with its receptors which consists of two IL-10R1 and two IL-10R2 molecules and the receptors so activated, trigger several signaling pathways which include the Jak1-Tyk2/Stat3 pathway [8].
IFNγ inhibits the proliferation of Th2 cells and enhances the proliferation of activated B cells. IFN-γ is expressed by NK cells and is involved in tumor immune surveillance through cytostatic and cytotoxic mechanisms [9]. It could have either pro or anti tumorigenic activity depending on cellular or molecular microenvironment of the tumor [9] [10]. It also inhibits tumor development by providing protection through lymphocytes and by immunoediting of the cancer cell phenotype [7]. Due to the tumor-sculpting roles of immunity, IFN γ is been proven to be involved in cancer immunoediting and not cancer immunosurveilance [9].
Extracts of Gongronema latifolium (AEGL) has been reported to show anti-inflammatory and antioxidant activities [11]. This study is aimed at investigating the anti-inflammatory properties of AEGL through its effect on the expression of IFNγ, and IL-10; and to trace the cellular sources of these cytokines through the expression of CD3, and CD56 in rabbits.

Experimental Animals
Twenty cross-bred male rabbits with an average weight range of 1. . Two weeks prior to the commencement of the study, the rabbits which were in good health were kept in the cages and no drugs were administered. The housing had a 12-hour light/dark cycle, a relative humidity between 36% -40%, and a temperature of 28˚C ± 2˚C. The study rabbits were administered roughage, commercial grower pelleted feed, and water ad libitum.
The design of the experiment and handling of the rabbits was in accordance with international standard [12].

Preparation of an Aqueous Extract of Gongronema latifolium
The leaves of the edible plant, Gongronema latifolium were obtained from a lo-

Experimental Design
The rabbits were separated into five groups of four rabbits each and treated as follows: Group 1 is the Untreated Control (with no extract and no multivitamins); Group 2 is the treated Control (administered or treated with 0.33 ml of Multivitamins (EMVITE ® Multivitamin Syrup) per kg body weight); Group 3 is treated with 200 mg/kg of AEGL; Group 4 is treated with 400 mg/kg of AEGL; and Group 5 is treated with 600 mg/kg of AEGL. Animals were weighed weekly and different doses of the aqueous extract of the plant were administered to the rabbits daily by intubation for 28 days.

Sample Collection, Preparation and Testing
Blood samples were collected from the lateral saphenous and ear veins of the rabbits at day 0, 7, 14, 21, and 28 of the administration of the AEGL. The plasma was obtained after centrifuging the blood at 2000 rpm for 10 minutes with Eppendorf Centrifuge 5702 (Eppendorf AG 22331, Hamburg, Germany).
The study samples and the IFNγ, Il-10 and CD56 ELISA kits (Elabscience, China) were removed from the refrigerator and kept at room temperature for 20 minutes before the commencement of the experiment. 30 ml of Concentrated Wash Buffer (Elabscience, China) was diluted with 720 ml of deionized water (dilution factor of 25). 1 ml of Reference Standard & Sample Diluent (Elabscience, China) was added to the Standard (Elabscience, China) which had already been centrifuged for 1 minute. This standard working solution (1000 pg/ml) was allowed to stand for 10 minutes and diluted into different concentrations. For the dilution, the Reference Standard & Sample Diluent (Elabscience, China) was used to obtain 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 0 pg/ml. To produce a Biotinylated detection Antibody (Elabscience, China) working solution, the Concentrated Biotinylated Detection Antibody (Elabscience, China) was centrifuged and diluted by a factor of 100 using the Biotinylated Detection Antibody Diluent (Elabscience, China). Also, to produce the Concentrated HRP Conjugate working solution (Elabscience, China), the HRP Conjugate (Elabscience, China) was diluted by a factor of 100 using the HRP conjugate Diluent (Elabscience, China). To each well, 100 μL of sample was added and incubated (DNP-9082 Laboratory Incubator) at 37˚C for 90 minutes. The liquid was removed from the wells, 100 μL of Biotinylated Detection antibody (Elabscience, China) was added, and incubated at 37˚C for 60 minutes. The solution was aspirated, and wells were washed thrice. 100 μL of HRP Conjugate (Elabscience, China) was added to the wells, incubated at 37˚C for 30 min, removed, and washed five times. 90 μL of Substrate Reagent (Elabscience, China) was added to the wells, incubated at 37˚C for 15 min, and 50 μL of Stop Solution (Elabscience, China) was added. At 450 nm wavelength, the optical density of the solution was read immediately with the GF-N3000 Microplate reader, (England) and the results calculated.
The CD3 kit (Bioassay Technology Laboratory, China) was removed from the refrigerator and kept at room temperature for 20 minutes before use. To produce 640 ng/ml standard stock solutions, 120 μL of Standard diluent (Bioassay Technology Laboratory, China) was added to 120 μL of standard (1280 ng/ml) (Bioassay Technology Laboratory, China). The solution was allowed to stand for 15 minutes before it was serially diluted. 320, 160, 80 and 40 ng/ml solutions were produced. Also, 20 ml of wash buffer concentrate (Bioassay Technology Laboratory, China) was added to 480 ml of wash buffer (Bioassay Technology Laboratory, China). 50 μL of standard solution (Bioassay Technology Laboratory, China) was added into the standard well. 10 μL anti-CD3 antibodies (Bioassay Technology Laboratory, China) were added to 40 μL of sample in the sample wells. To both sample and standard wells, 50 μL of streptuavidin HRP (Bioassay Technology Laboratory, China) was added. The plate was agitated for thorough mixing, covered, and incubated for 60 minutes at 37˚C. With the buffer solution, the plate was washed five times and blotted with paper towel. To each well, 50 μL of Substrate Solution A (Bioassay Technology Laboratory, China) and 50 μL of Substrate Solution B (Bioassay Technology Laboratory, China) were added. The plate was incubated for 10 min at 37˚C. 50 μL of Stop solution (Bioassay Technology Laboratory, China) was added to each well. The optical density read immediately using a microplate reader set at 450 nm wavelength.

Statistical Analyses
Data collected were analyzed using SPSS version 20 and subjected to one-way Analysis of Variance (ANOVA) in a completely randomized design. Significant means were separated. Using Duncan multiple range Test was and a P value less than 0.05 was considered significant.

Results
IFNγ: With respect to the effect of dosage of AEGL, there were significant differences (P < 0.05) in IFN γ levels amongst the treatment groups ( Figure 1).
The IFN-γ levels (574.25 pg/ml) in rabbits in Group 4 (administered 400 mg/kg AEGL) were higher (P < 0.05) than those of the control and other treatment groups. The effect of time was significant (P < 0.05) on IFN-γ levels declining from 621.20 pg/ml (baseline) to 306.56 pg/ml (day 28).
The trend as seen in Group 1 reveals an undulating trend as IFN γ levels de-     Figure 2 also reveals that highest (P < 0.05) baseline values of 395 pg/ml were recorded in Group 4. A significant reduction in IL10 activity over a 14-day period suggests that the greatest IL10inhibitory activity occurred at 400 mg/kg of AEGL when administered for 14 days. At 400 mg/kg, AEGL showed a stronger IL10-inhibitory activity than 0.33 ml/kg of Multivitamins (Group 2) whose activity only lasted for 7 days.
CD3: The effect of AEGL dosage on CD3 was not significant (P > 0.05) as seen in Figure 3. The effect of time was significant (P < 0.05) on CD3 levels declining from 884.60 pg/ml (baseline) to 51.16 pg/ml (day 21) The trend in Figure 3 reveals that the baseline values for CD3 in Group 3 (1259.8 pg/ml), Group 4 (1065 pg/ml) and Group 5 (1248.5 pg/ml) were comparably higher (P < 0.01) than those of other treatment groups. A decline (P < 0.01) in CD3 levels was seen from day 0 to 7 in groups 2, 3, 4 and 5 until Day 14 .CD3 levels in groups 2 and 3 remained undetectable from days 14 to 21. All groups showed recovery from day 21. Group 1 showed an opposite trend to all the other groups till day 21.
CD56: The effect of AEGL dosage on CD56 was not significant (P > 0.05) as shown in Figure 4. The effect of time was significant (P < 0.05) on CD56 levels   Figure 4 shows that the highest CD56-inhibition is at 400 mg/kg.
Trend seen showed a marked drop (P < 0.01) in CD56 levels from day 0 to 7 in all treatment groups. This was successively followed by an increase in cytokine levels (P < 0.01) in groups 1, 2, 3 and 4 from days 7 to 14. Days 14 -21 was characterized by a decline in CD56 levels while days 21 to 28 was seen in groups 1, 3, 4 and 5.

Discussion
IFNγ: Certain bioactive compounds contained in Gongronema latifolium have anti-inflammatory and antioxidant activities [11]. Like several other plant extracts [14] [15], AEGL can be used for the treatment of inflammation as they have been proven to decrease IFN-γ activity. The down regulation of the expression of TNF-α/IFN-γ through the blockade of several pathways is associated with the inhibition of ERK1/2 phosphorylation [13] [16].
It has been shown that many plants with anti-inflammatory effect could also some antibacterial properties [17] [18]. The roughages taken by Group1, Tridax pubscens and the multivitamins taken by Group 2 could also reduce IFN γ levels by their anti-inflammatory activity [19]. An upward significant (P < 0.05) trend in seen Group 3 from Days 7 and 14 is suggestive of an NKC-derived IFN γ production. Like bitter gourd (Momordica charantia) juice and the 70% hydromethanolic extract of Dioscorea alata, AEGL may also show pro-inflammatory activity by increasing IFN γ production [20]. IFNγ produced by NKC has been shown to have cytotoxic activity [21].
Over the 28-day period, IFNγ production is most significantly (P < 0.05) altered in Group 4 (400 mg/kg of AEGL). IFNγ levels reduced from 1119 pg/ml to 332 pg/ml in a 21-day period showing the greatest inhibitory activity. This suggests a maximum effective dose of 400 mg/kg of AEGL administered at 21 days.
High baseline values for Group 3 and 4 might be suggestive of a pre-existing inflammatory state where macrophages are activated which in turn trigger the production of Th1-derived IFNγ [22].
IL-10: Results suggests that AEGL is a natural inhibitor of IL10. In a similar fashion, Rituximab, a chimeric mouse antihuman CD20 antibody, inhibits IL10 resulting in the down regulation of bcl-2 and sensitization of B-cell non-Hodgkin's lymphoma to apoptosis caused by chemotherapeutic drugs [23]. IL10 is an immunosuppressive cytokine which directly affect NKC count. Elevated IL10 levels in a tumor microenvironment, suppresses IL12 which is the main stimulator of NKC production and it also down-regulates the expression TNF, and IFN-γ [23]. On the contrary, with low IL10 levels, IL12 is increased and this increases NKC cytotoxicity [24]. CD3: CD3 is an important and highly specific immunohistochemical marker for all subtypes of T-cells including immature T cells like pro-thymocytes and thymocytes. It is also marker for T cell lymphomas (differentiating them from B cell lymphomas) and leukemia [25] [26]. CD3 binds with subunits of TCR (alpha/beta ligand binding subunits) to form a complex that triggers signal transduction. Ligand binding with this complex induces conformational change of CD3 and recruits Nck adaptor protein [27]. The CD3 result shows the treated control and other treated groups caused the inhibition of CD3 T-cell proliferation. This might be due to the presence of common compounds such as Vitamin D. 1, 25-dihydroxyvitamin D3 which has been shown to inhibit the proliferation of T lymphocytes [27] [28]. CD56: is neural cell adhesion molecule. It is a typical example of phenotypic marker of natural killer cells and so cytotoxicity. The 400 mg/kg AEGL had the most significant up-regulatory effects on the marker (P < 0.05). The does may serve to for the detection of cytotoxicity.
Though known for its remarkable antioxidant and NKC stimulatory properties, the administration of 0.33 ml of multivitamins per kg live weight did not show enough potency as compared with 400 mg/kg of AEGL. High baseline values in groups 3 and 4 could be suggestive of an inflammatory process. Group 3 showed the highest inhibition of T cell activation while Group 4 showed the highest NK cell activity. However, it is important to note that the results and the interpretations may have been negatively affected by several factors which include the complex cytokine network interactions, nature of cytokines, genetic differences, experimental methodology and sample related issues [29] [30].
Established Facts:  Gongronema latifolium is a perennial edible plant with soft and pliable stem  Medicinal plants, including Gongronema latifolium, have anti-inflammatory activity  Medicinal plants have been used from the ancient times for human health benefits Highlights of the findings and novelties:  At 400 mg/kg for 21 days in rabbits, AEGL showed very strong anti-inflammatory activity against all the cytokines and cell surface markers  The molecular mechanism is through the expression of IFNγ and IL-10 cytokines via the expression of CD3 and CD56 cellular sources  Administration of 0.33 ml of multivitamins per kg live weight did not show enough potency as compared with 400 mg/kg of AEGL

Conclusion
In general, the production of all the cytokines was affected in time while IFN-γ levels increased in a dose-dependent manner following the administration of AEGL. IFNγ showed significant anti-inflammatory and pro-inflammatory activities because of their pleiotropic nature. The rabbits that received 400 mg/kg for all the cytokines exhibited significant activity which is suggestive of the maximum effective dose for AEGL (21 days for IFNγ and 14 days for IL10).