Stimulation of THP-1cells with LPs for stimulating the production of TNF-alpha
INTRODUCTION:
Tumor necrosis factor alpha (TNF-α) is plays a crucial role in acute infectious and inflammatory diseases. The exposure of any antigen in the body triggers the activation of various immune cells in the body e.g. monocytes and macrophages to eliminate the antigen from the body (Beutler et al., 2008). LPS is the endotoxin of gram negative bacteria which is a potential activator of immune cells. When activated by any bacterial antigen these differentiated immune cells start producing inflammatory cytokines. The role of TNF-α in acute bacterial infection is very significant in mediating signals for inflammatory cascades (Arditi et al., 1990). Use of anti-TNF-α therapies has been widely employed in the treatment and cure of such inflammatory and infectious diseases (Van Dullemen et al., 1995).
Many complications or different phenotypes of the inflammatory and infectious diseases can develop due to variation in the level of TNF-α produced after stimulus of any antigen. Low concentration of TNF-α produced after stimulation with a bacterial endotoxin especially LPS has been attributed with high risk of fatal meningococcal diseases (Westendorp et al., 1997). The reason behind expression of different level of TNF-α might be due to genetic background. Many variants of the TNF-α gene has been described (Wilson et al., 1993). .
Other than genetic background, expression of different concentrations of TNF-α has been attributed by the level of CD14 (clusters of differentiation) receptors in immune cells especially monocytes and macrophages (Ziegler-Heitbrock and Ulevitch, 1993). A direct co-relation between the number of white blood cells, degree of expression of CD-14 receptors on immune cells, production of other cytokines, and its soluble concentrations has been established with the amount and concentration of TNF-α produced (Groote, 1998).
Lipopolysaccharide is an important part of the gram negative cell wall that acts as endotoxin (Lüderitz et al., 1982). LPS is composed of side chain made up to different sugar groups and lipid core. The core region of the sugar side chain varies from species to species and the outermost region of the chain is the base of O-antigen variations (Lüderitz et al., 1966). The endotoxin property of LPS is mainly exhibited by the lipid A region (9). The LPS of all the bacteria of enterobacteriaceae family possess maximum pro-inflammatory properties (Raetz, 1993). LPS is one of the most powerful stimuli for the monocytes activation. Monocytes start to produce proinflammatory cytokines such as IL-6, IL-1, and TNF-α when they are triggered by LPS which is followed by IL-10 production ( Westendorp et al., 1997, Valeri and Raffatellu, 2016).
TNF-α is a necessary component to modulate the non-specific immunity which involves the prompt host defense against antigens before the adaptive immunity activation (Vujanovic, 2011). Macrophages produce TNF-α in the reaction of membrane-bounded pattern-recognition molecules activation like TLR activation which identifies usual products present on cell surface of bacteria such as LPS. Many other types of structural cells like smooth muscle cells, epithelium and fibroblast and proinflammatory cells like eosinophils, mast cells, neutrophils, CD4 + cells, dendritic cells, B cells, and monocytes also produce TNF-α (Strieter et al., 1993).
This study was designed to check expression of interferon alpha an inflammatory cytokine. For this human monocyte cell lines were cultured into the lab. The monocytes were differentiated into macrophages by MPA. The differentiated cells were then challenged with different concentrations of bacterial endotoxin (LPS) to check the concentration of the TNF-α produced. The commercially available uncoated sandwich ELISA kit was used for this purpose.
OBJECTIVES:
The study was carried out for the following purposes:
- To investigate the potential of LPS (lipopolysaccharide) to stimulate the production of inflammatory cytokine, tumor necrosis factor alpha (TNF-α).
- To check the expressions of TNF-α in TNH-1 cells when challenged with different concentrations of LPS.
METHODOLOGY:
- THP-1 cells:
THP-1 cells suspension was prepared in cell culture essential medium. The cells were examined under microscope and counted for cell number. After counting, the cells were centrifuged and supernatant was discarded for sub-culturing of the cells. The supernatant was discarded and cells were re-suspended in RPMI medium and final concentration of 5×105 cells /ml was prepared. This suspension was prepared in the final volume of 20ml of the cell culture medium. RPMI medium used for culturing the cells was supplemented with L-Glutamine (10,000 units /ml; 1% in medium), Penicillin/streptomycin solution (10,000 units/ml; to prevent bacterial contamination), amphotericin B – to prevent fungal infection and foetal calf serum (FCS) 10%.
- Differentiation of THP-1 cells with Phorbol -12 myristate-13 acetate (PMA):
The monocytes in THP-1 cells were differentiated into macrophages by adding PMS (phorbol 12-myristate 12 acetate). The final concentration of 0.5ng/ml of PMA was prepared from the stock solution. The cultured cells were centrifuged and re-suspended into the culture medium and dispensed in 24 wells tissue culture plate. A total of 10ul of PMS (phorbol 12-myristate 13-acetate) was added to each well. The tissue culture plate was the incubated at 37˚C at 5% CO2 for 72 hours.
- Resting Cells:
After incubation, differentiated cells were semi adhered to the wells of the tissue culture plate. All the wells were examined critically under the microscope for any change in the cell morphology and cell differentiation was confirmed. The supernatant from the wells was removed carefully and freshly prepared media was added into the cells without disturbing them.
- LPS Stimulation of THP-1 Cells:
Different concentrations of LPS 0ng/ml, 1ng/ml, 10ng/ml, 50ng/ml, and 100ng/1ml were prepared from the stock solution provided. These dilutions were prepared in RPMI medium that was supplemented with 10% foetal calf serum. Different concentrations of LPS prepared were added to cells cultures in tissue culture plate to stimulate the production of TNF-α. Each dilution was added in three replicates into the cultured cell to check the dose dependent expression of TNF-α production. The cells were then incubated at 37˚C at 5% CO2 for 24 hours.
- ELISA:
After incubation, the supernatant from the cultured cells was harvested and expression of TNF-α was determined via commercially available Human TNF alpha Uncoated ELISA kit according to instruction manual. The optical density values and the concentration measured were noted.
RESULTS
Cell culture:
The THP-1 cells were cultured in RPMI medium containing 10% fecal calf serum, antibiotics and antifungals. The cells were cultured in suspension form. The cell counting was done via hematocrit method and the final concentration of 5×105/ml was prepared in 20ml of the RPMI medium.
Differentiation of THP-1 cells: THP-1 cells are human monocytes that were differentiated into macrophages addition of MPA (phorbal -12 myristate -13). A range of different concentrations (0 to 100ng/ml) were used to challenge cultured cells for their differentiation into macrophages. The cells were dispensed into tissue culture plate before addition of the MPA. 24 hour incubation under ideal culturing conditions was given to the cells after their exposure to various concentrations of prepared MPA. Microscopic examination revealed that all the cells were semi-adherent to the surface of the wells, and were completely differentiated into macrophages.
LPS stimulation and TNF-α assessment via ELISA:
After the cells were differentiated into macrophages, they were challenged by different concentration of LPS for the production of tumour necrosis factor alpha. Each concentration prepared was run in three replicates to avoid human error. The concentrations of LPS prepared were 0, 1, 10, 20/50, and 100ng/ml. The cells were the incubated for 6 hours under ideal culturing conditions. After incubation, supernatant was harvested from the wells for determining the dose dependent expression of TNF-α. Commercially available ELISA kit for the detection of TNF-α was used for this purpose. OD values measured for different concentrations of the endotoxin were noted down and the corresponding concentration of TNF-α produced for each LPS concentration were also measure via Gene 5 software attached to the ELISA reader.
Standard curve for TNF-α:
A standard curve for different concentrations of TNF-α was generated. The standard of TNF-α was provided with the ELISA kit. The concentration of the stock standard solution was 500pg/ml. 2-fold serial dilutions (starting from 500pg/ml to 0pg/ml) of TNF-α standard were prepared to estimate the corresponding OD values to make a standard curve. Each dilution was run in three replicates and OD values were measured. BioTek Gen5 data analysis software for calculating the concentration of TNF-α from OD values was also used. The concentration calculated by the software was cross checked for their matching with serially diluted standard TNF-α concentration against respective OD. The results obtained are summarized in the table below:
| TNF-α standard (pg/ml) | OD- replicate 1 | OD-replicate 2 | OD-replicate 3 | Average OD | Concentration (pg/ml) |
| 0 | 0.094 | 0.1 | 0.086 | 0.093333 | 0.346 |
| 7.8 | 0.156 | 0.154 | 0.152 | 0.154 | 8.403667 |
| 15.6 | 0.212 | 0.216 | 0.214 | 0.214 | 15.51967 |
| 31.25 | 0.359 | 0.354 | 0.343 | 0.352 | 30.85567 |
| 62.5 | 0.637 | 0.647 | 0.617 | 0.633667 | 61.459 |
| 125 | 1.254 | 1.172 | 1.155 | 1.193667 | 127.034 |
| 250 | 1.951 | 2.091 | 1.985 | 2.009 | 248.5847 |
| 500 | 2.97 | 3.101 | 3.06 | 3.043667 | 501.0703 |
Table 1: Optical density (OD) value of different concentrations of TNF-α standard
The table shows the OD values of different concentrations of TNF-α standards and their concentration determined via BioTek Gen5 data analysis software. A direct relation between OD value and concentration is obvious. The dose dependent direct relation between TNF-α and OD values was noted and a standard curve was prepared. Highest OD value was observed was 3.02 at highest concentration (500pg/ml) of TNF-α, and lowest OD value 0.09 was measured highest dilution of TNF-α (0pg/ml).
Figur 1: TNF-α standard curve .The OD values (measured at 450nm) for different concentrations of TNF-α has been shown.
Challenging THP-1 cells with LPS:
Different concentrations of LPS were added in cultured THP-1 cells in three replicates, and incubated for about 24 hours to stimulate the production of TNF-α. The cultured supernatant was harvested and TNF-α expression was measured via sandwich ELISA. It was also quantitated via BioTek Gen5 data analysis software. The OD values for TNF-α produces against different concentrations of LPS are summarised in the table below along with the concentration measured via data analysis software.
| LPS (ng/ml) | Average OD value | Average concentration of TNF-α (pg/ml) | Average conc. of replicate 2 | Average conc. of replicate 3 |
| 0 | 0.141 | 6.762667 | 7.253333 | 11.366 |
| 1 | 2.371667 | 320.4563 | 310.8717 | 525 |
| 10 | 2.676333 | 392.1347 | 394.7777 | 525 |
| 20/50 | 2.606667 | 375.636 | 478.8187 | 525 |
| 100 | 2.379333 | 321.0627 | 525 | 525 |
| 10d | 1.079667 | 113.8237 | 89.04233 | 246.4203 |
| 50d | 1.582667 | 180.1797 | 107.2517 | 269.7453 |
| 100d | 1.546667 | 176.2007 | 179.6383 | 471.9013 |
Table 2: The table shows OD values of TNF-α obtained at different concentration of LPS
A direct dose dependent relation between different concentrations of LPS and TNF-α was observed. Challenging the cells with an increasing concentration of LPS resulted in increased concentration of TNF-α produced. Highest OD value of TNF-α was noted at 50ng/ml dilution of the LPS. The optical density values observed at 0ng/ml, 1ng/ml, and 10ng/ml were 0.14, 2.37 and 2.67 respectively. The OD values then started decreasing with increasing concentration of LPS. The optical density value measured at 100ng/ml of the LPS was 1.54 (Table 2).
Figure 2: Graphical representation of LPS dose dependent expression of TNF-α. The increasing concentration of TNF-α with increasing level of LPS is indicative up to 10ng/ml. Then pattern is of got changed and concentration of TNF-α started decreasing with increasing concentration of LPS.
The supernatant harvested from the incubated cells was also prepared in different dilutions (10d, 50d, and 100d) for checking the dilution dependent expression of the TNF-α. Same pattern of first increasing then decreasing concentration of TNF-α was observed. The highest concentration of TNF-α produced was observed where supernatant was diluted 50th times.
Table 3: graphical representation of the concentration of TNF-α produced at different dilutions of supernatant
Statistical Analysis: The data obtained was analysed statistically by t-test. The results were found to be statistically significant.
Discussion:
Lipopolysaccharide (LPS) is very potential activator of macrophages and monocytes. It is an integral part of gram negative bacteria. In acute inflammatory and infectious diseases it stimulates the synthesis of several inflammatory cytokines from immune cells to launch immunity against the bacterial endotoxin. It can provoke differentiation of immune cells both in vivo and in vitro. In recent study THP-1 cells were differentiated into macrophages with by PMA (Phorbol -12 myristate-13 acetate, and then these cells were challenge with different concentrations of LPS for estimating the production of TNF-α. The TNF-α expressed due to different concentration of the bacterial endotoxin (LPS) were assessed from the OD values and also quantified via a software. A dose dependent direct relation between LPS stimulation and TNF-α was measured up to 10ng/ml concentration of LPS. Treatment with 0, 1, and 10ng/ml of LPS resulted in increasing level of TNF-α. But TNF-α expression was reduced when concentration of LPS was further increased. The results indicate that LPS is a potential activator of TNF-α production, an inflammatory cytokine that recruits other inflammatory cells to the infection site to combat against it. One of the possible reasons of decreased concentration of TNF-α after certain concentration of LPS stimulus might be the production of other inflammatory cytokines upon stimulus of bacterial LPS after a certain concentration. The other cytokines e.g. IL-6, IL-10 etc produced might have a negative feedback on the production of TNF-α. Liu et al. 2018 demonstrated same pattern of expression of TNF-α when human THP-1 cells were treated with different concentrations of LPS at different time interval. Dose dependent increasing concentration of TNF-α was measured with increasing concentration of LPS. (Liu et al., 2018).
Perez et al., 1995 have also demonstrated the activation of TNF-α expression in THP-1 cells and other cell lines. The cells were treated with different concentrations of LPS of Helicobactor pylori and a direct relation between TNF-α expression and LPS exposure was observed with great precision. (Pérez-Pérez et al., 1995).
Louis et al., 1998 has demonstrated different reasons for variations in the expression of the TNF-α. He stated that there could be several factors affecting the production and concentration of TNF-α. These could be genetic background of the individuals, expression of the CD14 receptors on immune cells and production of other cytokines masking the synthesis of TNF-α. The last is of great significance explaining the reason of weird finding the present study.
Conclusion:
The study indicates that human monocytes when challenged to a bacterial endotoxin (lipopolysaccharide) are triggered to produce different inflammatory cytokines, tumor necrosis factor alpha (TNF-α) is amongst one of them. The present study indicated dose dependent increasing relation between TNF-α expression and different concentrations of LPS. Up to certain levels, the relation between LPS and TN-α is direct, but concentration increasing than the thresh hold level masked the expression of TNF-α in both diluted and un-diluted cell culture supernatant/. This might be due to the reason that production of other cytokines being may have a negative feedback on the expression of TNF-α. It is also suggestive of the fact that concentration of LPS exposure is highly associated with the onset of synthesis and expression of different cytokines.
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