dissertation 1

Fenugreek extract in the regulation of the inflammatory response and polarization in human macrophages

By Arrandhev Singh Kambo ST20034270

Supervised by Dr. Keith Morris

Arrandhev Singh Kambo st20034270

Acknowledgements

I would like to thank Stephen Potter, Richard Roll ands and Sam Hooper for their technical assistance throughout the course of the project. I would like to also thank Nurudeen Hussan for providing the methanolic extraction of Trigonella foenum-graecum protocol and the preparation of the cytokines before all experiments.


1

Declaration

Statement 1

This work has not previously been accepted in substance for any degree and is not being

concurrently submitted in candidature for any degree.

Signed: ……………………….……………………… (Candidate)

Dated: ……………………....

Statement 2

This dissertation is the result of my own investigations, except where otherwise stated. Where

corrections services have been used, the extent and nature of the correction is clearly marked

in a footnote. Other sources are acknowledges by footnotes giving explicit references. A

bibliography is appended.

Signed: ……………………………………………… (Candidate)

Dated: ……………………....

Statement 3

I hereby give consent for my dissertation, if accepted to be available for photocopying and for

inter library loan, and for the title and summary to be made available to outside organizations.

Signed: ……………………….……………………… (Candidate)

Dated: ……………………....


2

Abstract

Flavonoids are naturally-derived compounds that are present in a wide range of fruits and

vegetables. Previous studies have demonstrated that flavonoids produce anti-inflammatory

activity in vitro and in vivo, which could be used to inhibit the progression of a number of

inflammatory diseases such as diabetes mellitus, asthma or atherosclerosis. Certain

flavonoids express their anti-inflammatory effects by modulating specific mediators of the

inflammatory process such as cytokines, chemokines or markers of inflammation. Previous

research has linked these mediators such as CD86 and CD163 markers in polarization of

macrophages into a M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotype. The

purpose of this study was to investigate the capabilities of Trigonella foenum-graecum, also

known as fenugreek in regulating the macrophage polarization process with the use of a THP-

1 cell model. THP-1 macrophages (MΦ) were treated with two different concentrations of

fenugreek extract in the presence of cytokines IL4 and IL10, which were used as positive

controls. Fluorochrome conjugated CD86 (M1 marker) and CD163 (M2 marker) expression

was analysed to measure the effect of fenugreek extract on THP-1 MΦ. The effect of

fenugreek on these markers would give evidence as to if flavonoids are capable of regulating

the polarization process. This study found that IL4 and IL10 produced an effect on both

markers as a result of both these cytokines being involved in the inflammatory response. This

study found that fenugreek had upregulated the CD86 marker however it downregulated

CD163. Fenugreek had caused macrophage polarization within CD86 bound macrophages,

however this polarization shifted the M1 phenotype to an M4 phenotype, the reason for this

would require further research. The effect fenugreek produced on the CD163 marker could be

due to the number of subclasses in the M2 phenotype, this make it more difficult to measure

the expression within this type of macrophage.


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1.Introduction

Inflammation is a fundamental human reaction to disease and injury, characterized by specific signals – pain, heat, redness, swelling and potential loss of function. The inflammatory response is an important part of the body’s defence system, acting as an alarm to notify immune cells they are under attack. External stimuli trigger the inflammatory response causing damaged cells to excrete a number of chemical signals, including histamine, prostaglandins and cytokines.

Chronic-low grade inflammation is involved in the pathogenesis of metabolic diseases such as obesity derived diabetes mellitus and atherosclerosis [1]. Macrophages play a key role in inflammation and are capable of responding to a range of environmental signals by secretion of inflammatory mediators and phagocytosis. The diversity and overlapping of signal cues generates a spectrum of macrophage phenotypes, classified as classically activated pathway (M1) and alternatively activated pathway (M2) [2-4}. Release of specific cytokines will cause a polarization shift, whereby mononuclear phagocytes in response to environmental signals. For example, interferon-gamma (IFN-γ) and bacterial lipopolysaccharide (LPS) are pro-inflammatory stimuli that activate the M1 sub-type causing the release of proinflammatory cytokines including IL-1, IL-6 and TNF-α. Conversely anti-inflammatory cytokines such as IL-4 and IL-13, produced by Th-2 lymphocytes promote M2 macrophage development, aiding immune regulation and tissue repair [5]. M2 macrophages release anti-inflammatory cytokines such as. IL-10 and IL-1ra (interleukin-1 receptor antagonist) [12]. Accumulation of data from previous journals have indicated an imbalance of M1 and M2 population particularly a chronic production of the M1 sub-type can cause tissue damage [6-8] [10]. Further research demonstrated the importance of macrophage polarization in the pathogenesis of a diverse range of inflammatory disorders, but also seen to contribute to neurological diseases such as multiple sclerosis [8-10]. In this study a human monocytic leukemia cell line known as THP-1 was used. THP-1 has been used extensively in studies investigating the function and regulation of monocytes and macrophages in response to inflammatory stimuli.


4

Fenugreek seeds or Trigonella foenum graecum (Tfg) are classified as complementary and alternative medicine (CAM), commonly used in Indian, Chinese and Middle-eastern medicine as a substitute to western-forms of treatment. It has been successfully implicated in the management of diabetes and some forms of cancer [11]. A meta-analysis of multiple trials that used Tfg seeds demonstrated its potential in lowering blood glucose in diabetic participants [11]. The aim of this study was to investigate the effects of fenugreek extract (FE) in regulating the polarization shift between M1 and M2 state. Specifically, this study compared the expression of CD86 marker to CD163 marker using THP-1 cell model. The M2 marker CD163 is a hemoglobin scavenger-receptor specifically expressed on the surface of macrophages and monocytes, its general purpose is the clearance of hemoglobin [14]. This marker is shed by activated monocytes/macrophages, via proteolytic cleavage at the outer surface of cell [15]. The M1 marker CD86 is an antigen presenting cell that binds to CD28 (a T cell marker) to initiate a stimulatory cascade [16], CD86 is expressed on both monocytes and macrophages. It is found primarily produced by alveolar-based basophils [17-20] in response to an allergic reaction, which promotes the upregulation of CD86 along with CD28 to initiate the pro-inflammatory response [17]. Two cytokines called IL4 and IL10 would be used as positive controls for this expression. Our hypothesis was that we expect fenugreek extract to significantly increase the expression of both markers, hence priming THP-1 macrophages to a M2 phenotype.


5

2. Methods & Materials:

2.1 THP-1 Propagation and Preparation

Figure 1: the well plate above displays how cell culture was laded out for this pre-experimental step.

THP-1 cells were propagated in RPMI 1640 supplemented with 10% Foetal Bovine Serum,

1mM sodium pyruvate, 2mM glutamine and 1% of non-essential amino acids (all

components supplied by Thermo Fisher Scientific, UK). Cells were cultured at 37oC in a

humidified atmosphere with 5% CO2. THP-1 monocytes were passaged once growth reached

80% confluence (0.8-1.2 x 106 cells/ml) and were differentiated into macrophages (Mφ) with

100ng/mL phorbol-12-myristate-13-acetate; PMA (Thermo Fisher Scientific, UK). The

PMA-supplemented cells were added a 6 well culture plate and incubated for 24 hours in a

5% CO2 humidified environment. Following this, cells were washed in RPMI-1640, before

the addition of fresh media and incubated for 48 hours. PMA treated THP-1 cells where

added to each well in a 6 well plate culture. IL-4 was constituted using working

concentration of 20ng/mL in 100 ug/ml of stock concentration and IL-10, which was

constituted at a working concentration of 20ng/ml. Both interleukin acted as positive control

variables and where diluted down to 4uL in 10ml and 2uL in 10ml of DPBS. Both CD86 and

CD163 were dependent variables in this experiment. Isotype controls (20ul dilution in 400ul

FACs buffer) were the negative control.


1 2 3

4 5 6

6

Well number Contents

1 Isotype only

2 Isotype only

3 CD163/CD86 only

4 CD163/CD86 only

5 CD163/CD86 + IL4

6 CD163/CD86 + IL4

2.2 Sub-culturing Techniques for Adherent Cells

Proliferation of cells continue until the surface area of each well had been enveloped by

adherent M1 macrophages. During this plateau cells underwent subculturing to prevent

apoptosis. Multiple methods where utilised on three 6 well culture-plates to determine which

would yield the maximum percentage of cells viable in suspension using the flow cytometer.

Each plate was treated with said agent to identify its capabilities in detaching THP-1

macrophages from substratum, each plate was incubated for approximately 2-10 minutes at

37oC. Detachment of cells was confirmed visually by examination under an inverted

microscope (Olympus 2k). To remove any remaining cells gentle shaking of the plates was

performed and media was re-homogenized using simple pipette technique for a further 2-5

minutes (varied time frame dependent on amount of cell-cell contact seen in each well). Flow

cytometer (BD Acurri Flow Cytometer C6) was utilized to compare each technique by

tallying total percentage of cells viable against the number of cell death induced. 10mM

EDTA diluted in DPBS was deemed the optimal, as it produced high percentage cell viability

against induced cell death.

2.3 Addition of Antibody to Culture

Once cells were detached, the suspension was transferred from each well to 12x75 mm2

falcon round bottomed tubes (Fisher Scientific, UK) and washed twice by centrifugation at

250RCF for 5 minutes and resuspended in 1ml of ice cold FAC’s buffer (DPBS, 3% v/v

foetal bovine serum and 0.1% w/v sodium azide: NaN3) to neutralize EDTA. Primary

fluorochrome-conjugated antibody and antibody isotype (APC anti-human CD163 & APC

mouse IgG1 Isotype, Biolegend, USA) were diluted using PMA stock (1mg/ml) to a final

concentration of 5ng/ml and transferred into FACs buffer in bulk to reduce sample variation.

Once diluted in FAC’s buffer 100uL of antibody/isotype was pipetted off and added to falcon

tubes. Tubes must be incubated on ice in the dark for 40 minutes to allow for optimization. At

20 minutes’, samples were vortexed, ensuring all antibodies/isotype bind to cells. Three

follow up washing procedures were performed (250RCF for 5 minutes) and FACs buffer

(1ml), after final wash 500ul of FACs buffer was added.


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2.4 Extraction Preparation of Fenugreek (as instructed by Mr. Nurudeen Hussan)

40g of dried fenugreek seeds (obtained from a commercial source) were washed with distilled

water and ground using a mixer into a fine powder. Powder was transported into a 100ml

flask containing 100% methanol for incubation in a rotary mixer overnight at 25oC. Solvent

was spun in centrifuge for 15 minutes and fractions were recovered. A rotary evaporator

(Bibby RE100, UK) evaporated excess methanol and air-free technique using nitrogen (N2)

dried-out the residue. The extract was dissolved in dimethyl sulfoxide (DMSO) and filtered

through 0.22um PVDF filter, stored at -20oC. Extract was thawed and reconstituted in fresh

media for any experiments.

2.5 Addition of Extract to Culture

Figure 2: Experiment Plate layout, including independent variable (FE) and dependent variable (CD163/CD86)

in each well.


1 2

3 4

5 6

1 2 3

4 5 6

7 8 9

10 11 12

8

Wells (no.) Contents within well (ug/ml)

1 Cells only

2-3 Isotype only

4-6 Cells+CD163

7-9 Cells+CD163+IL4

10-12 Cells+CD163+IL10

Wells (no.) Content within well (ug/ml)

1 Cells+CD163+FE Conc 1

2 Cells+CD163FE Conc 1





Figure 2 depicts the layout of the cell culture with the introduction of fenugreek extra).

Fenugreek extract (FE) was cultivated for this part of the protocol to create a comparison of

CD163 when exposed to varied concentrations of FE. The dilution process for each

concentration of fenugreek were as follows: FE concentration 1 used working concentration

of 30ug/ml in 37mg/ml of stock and diluted to 8.1uL in 10ml. FE concentration 2 used

working concentration of 60ug/ml in 37mg/ml of stock and diluted to 16.2uL in 10ml. The

two fenugreek concentrations were added to media in well-plates. Differentiated THP-1 cells

were seeded in 5ml of media in 12 well-plate, cultured alongside IL4 and IL10 and the other

a 6 well-plate cultured alongside fenugreek stimuli. Both plates where incubated in a 5% CO2

humidified atmosphere at 37oC for 48 hours before the addition of the primary fluorochrome

antibody/isotype.

2.6 Flow Cytometer analysis

After the well-plates were incubated on ice in the dark along with FE analysis is required to

understand the effects this variable has had on CD163 expression. Flow cytometry is a widely

used method for analyzing expression of cell surface receptors, also able to give accurate cell

viability percentages. Fluorochrome-conjugated CD163 and isotype were excited via flow

cytometry laser causing cells to radiate light which is filtered at multiple wavelengths,

allowing for sensors to detect fluorescence at your preferred wavelength. Measuring the

strength of the signal is also possible via sensors which convert the fluorescence into voltage

peaks. These voltage peaks will correlate directly to the intensity of the fluorescence for that

population of cells. Finally, the fluorescence intensity can be used to determine anomalies

within data via positioning of the peaks on the graph. A negative result is deemed so if peak

is closer to 0, meaning lack of staining, whereas a positive result is shifted to the right,

meaning a more intense fluorescence.


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3.0 Results

3.1 Determining the ideal detachment technique for THP-1 Mφ cell viability

To choose the most suitable detachment method for differentiated THP-1 cells a series of

duplicate plates were prepared to compare the three procedures considered for this

experiment: trypsin, cell scraping and EDTA. Trypsin an enzymatic agent that breaks down

the proteins that form between cells and plate surface. Trypsin and cell scraping techniques

where both deemed unsuitable, although figure 3.1 show high mean fluorescence for CD163

and CD86 markers the overall cell viability (%) was significantly reduced, due to

differentiated THP-1 Mφ remaining adhered. Subsequently, EDTA technique was the

preferred choice in the detachment protocol, a chelator that isolates Ca2+ and Mg2+ cations

required for adhesion. This method takes a longer time to incubate than the two previous

methods however, it was overall the most successful as detaching viable cells whilst retaining

the fluorescence of the markers expressed on each THP-1 cell.

cells sc

rape

cells EDTA

cells Tryp

sin

isotyp

e+163+ Scrape

isotyp

e+163+ EDTA

isotyp

e+163+Trypsin

cd163 + Scrape

cd163 + EDTA

cd163 + Trypsin

cd163 +IL4+Scra

pe

cd163+IL4+EDTA

cd163+IL4+Tryp

sin

0

1000

2000

3000

4000

5000

6000

Mea

n Fl

uore

scen

e In

tens

ity/F

L4A

Figure 3.1: Effect of detachment techniques Trypsin, EDTA and Scraping. A visibly higher expression of CD163 in differentiated THP-1 was seen in red. This expression increased further in presence of a M2 marker IL-4.


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Statistical Analysis

All data was presented as the mean ± standard deviation. One-way analysis of Variance (ANOVA) was conducted for all test sample comparisons. The statistical program, Minitab was used for statistical analysis. Statistical significance is represented in each legend as: P value equal to (=) or less than (<) 0.005; *. Error bars represent standard deviations of at least three replicates.

cells + EDTA

cells + Tryp

sin

cells + Scra

ping

isotyp

e + EDTA

isotyp

e + Trypsin

isotyp

e + Scrape

cd86 + EDTA

cd86 + Trypsin

cd86 + Scrape

cd86 +IL4 + EDTA

cd86 +IL4 + Tryp

sin

cd86 +IL4 + Scra

pe0

5000

10000

15000

20000

25000

30000

Mea

n Fl

uore

scen

e In

tens

ity/F

L4A

Figure 3.2: Effect of detachment methods on CD86 expression. Mean fluorescence expression of the CD86

marker was significantly higher when cells where exposed to EDTA, suggesting there is variable differences in

the performance of each agent depending on which marker is tested. In the future a potential improvement could

be a combination of Trypsin and EDTA in a diluted solution such as 0.025% trypsin solution diluted in EDTA.

3.2 Effect of organic fenugreek extract (FE) on CD163 Marker in THP-1 cell


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Initial experimental design was to implement a single dose concentration of FE (30ug/ml) to

all samples in attempts to analyse the compound’s effect on CD163 expression. An

upregulation within cells was seen when subjected to a low concentration of FE. CD163

coupled with IL4 and IL10 was expressed strongly, in comparison to the isotype and so clear

effect induced by FE is evident.

Isotype CD163+IL4 CD163+IL10 FE0.00

500.001000.001500.002000.002500.003000.003500.004000.004500.005000.00

*

*

*

Mea

n FL

uore

scen

ce In

tens

ity/F

L4A

Figure 3.3: Effect of FE on mean fluorescence intensity. THP-1 Mφ cells were treated with 30ug/ml FE for 24 hours. The difference between treated samples was determined by One-way ANOVA. (P value = <0.0001)

Although the initial data is encouraging there were limitations to this initial experiment, to

gain better comparison between data more variables were needed, this would require the

introduction of additional control variables. Additionally, low level of expression of the

marker was seen when 30ug/ml of FE was applied, to test the effects of FE varied

concentrations of FE are needed to see if this effect on THP-1 MΦ continues when exposed

to higher concentrations of the extract.

3.3 Effect of organic fenugreek extract (FE) on CD86 marker in THP-1 cell


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The same single dose concentration of FE was applied CD86, FE caused a notable

upregulation of the marker, this indicates that FE is successfully altering the macrophage

phenotype. However, higher expression of this marker when IL4 was applied to cells

(20,000~), this abnormal result was difficult to determine, it may have been due to human

error or false positive result. Additional experiments would therefore be required to

determine if the CD86+IL4 test sample result was a false positive or a true value.

Isotype CD86 + IL4 CD86 + IL10 FE 0

5,000

10,000

15,000

20,000

25,000

*

*

Mea

n Fl

uoro

scen

ce In

tens

ity /

FL2A

Figure 3.4: Effect of FE on CD86 expression via mean fluorescence intensity. THP-1 MΦ cells were treated

with 30ug/ml of FE. The difference between treated samples was determined by one-way ANOVA (P value =

0.005) CD86+FE result showed a clear reduction in fluorescence, this could be due fenugreek’s anti-

inflammatory properties.

Contamination was seen in 2/3 tested FE samples once 24-hour incubation had concluded.

This was as a result of a biological contaminant entering media, causing a lack of growth

within cell line and therefore prevent the testing of these samples. As a result of this

CD86+FE data was deemed as statistically insignificant, thus jeopardizing the validity of this

data. Therefore, FE+CD86 test would need to be repeated, as well as additional experiments

to include varied concentrations of FE.


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3.4 Comparing effect of fenugreek extract (FE) on THP-1 monocytes and

macrophages

Due to contamination error and the need for additional variable and varied concentrations of

FE improvements were made to the study. More control variables were incorporated and each

sample was carried out in triplicate. Two doses concentrations of FE (30ug/ml and 60ug/ml)

were applied to both experiments. Comparing results between the two cell types it’s evident

that fenugreek still produced an anti-inflammatory response in both however the degree of

effect is varied.

Cells Isotype CD163 CD163+IL-4 CD163+IL10 CD163+FE 1

CD163+FE 2

0.00

100.00

200.00

300.00

400.00

500.00

600.00

*

**

* *

Mea

n FL

uore

scen

ce In

tens

ity/F

L4A

Figure 3.5: Effect of FE on CD163 expression when adhered to monocytes via mean fluorescence intensity. All

cells were treated with two concentrations of FE (30ug/ml & 60ug/ml). The difference between treated samples

where determined using one-way ANOVA (P=<0.0001). Graph depicts a drop in CD163 expression when FE

was added, the drop in expression was identical in both concentrations.


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cells only isotype cd86 cd86 + IL4 CD86 +IL10 CD86 + FE conc 1

CD86 + FE conc 2

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0 *

*

*

**

Mea

n Fl

uore

scen

ce In

tens

ity/F

L2A

Figure 3.6: Effect of FE on CD86 expression when adhered to monocytes via mean fluorescence intensity. All

cells were treated with two concentrations of FE (30ug/ml & 60ug/ml). The difference between treated samples

where determined using one-way ANOVA (P values = 0.005). Graph depicts a slight drop in the first FE

concentration, this dropped further as the concentration increased.

Introducing two concentrations of FE produced varied results, using an increased

concentration of FE (60ug/ml) did not yield higher expression. Both concentrations caused an

upregulation of CD86 expression, this increase suggests THP-1 macrophages are

experiencing a shift in polarization away from M1 phenotype. Monocytes treated with CD86

experienced an elevated expression in comparison to monocytes treated with CD163, this

pattern continued when differentiated THP-1 cells were treated with CD86. However,

concentration 1 was seen to cause a greater response by the marker than concentration 2. This

suggests that increasing dose of extract added to cells would not necessary create a greater

expression within markers and other variables linked to FE must be explored into increasing

the expression. The CD163 expression was significantly reduced when exposed to both

concentration this was an unexpected result which would require further analysis.


15

Cells Isotype CD163+IL4 CD163+IL10 CD163+FE 1 CD163+FE 20.00

500.00

1000.00

1500.00

2000.00

2500.00

*

*

**

Mea

n Fl

uore

scen

ce In

tens

ity/ F

L4A

Figure 3.7: Effect of FE on CD163 expression when adhered to M1 macrophages via mean fluorescence

intensity. All cells were treated with two concentrations of FE (30ug/ml & 60ug/ml). The difference between

treated samples using one-way ANOVA (P=<0.006). Graph above depicts a severe drop in expression when

CD163 adhered macrophages where exposed to both concentration of FE.

isotype cd86 cd86 +IL4 cd86 +IL10 CD86+FE 1 CD86+FE 20

2000

4000

6000

8000

10000

12000

14000

16000

18000

*

*

*

*

*

Mea

n Fl

uore

scen

ce In

tens

ity/F

L2A

Figure 3.8: Effect of CD86 expression when adhered to M1 macrophages via mean fluorescence intensity. All

cells were treated with two concentrations of FE (30ug/ml & 60ug/ml). the difference between treated samples

using one-way ANOVA (P values = 0.005). Graph above showed that CD86 treated with varied concentrations

of FE displayed only a slightly reduced expression in comparison to CD86 adhered macrophages without FE.


16

4.0 Discussion

The aim of this study was to investigate the ability of methanolic extract of, fenugreek seeds

that contained a wide range of polyphenolic compounds to regulate macrophage polarization

shift in THP-1 M1 macrophages (MΦ). This study specifically aimed to use known M1 &

M2 markers (CD86 and CD163) bound to differentiated THP-1 MΦ to monitor the level of

expression when fenugreek extract (FE) was added to both CD markers and to measure any

reaction elicited by IL4 and IL10 cytokines, which were the positive control variables.

Analysis was carried out using flow cytometry to measure fluorescence emitted by the

fluorochrome bound CD markers and to measure the cell viability (%) throughout all

experiments. Our predicted hypothesis was that FE would cause a polarization shift from M1

phenotype to a M2 phenotype by causing an upregulation of both markers.

4.1 Fenugreek Extract effect on CD163 Marker

There is a high expression of this marker in M2 macrophage phenotype, playing a major role

in dampening the pro-inflammatory response produced by the M1 macrophage phenotype.

Studies have spoken of the mechanism of CD163 in the regulation of inflammation, currently

there are two routes as to how this marker produces an anti-inflammatory effect. First, CD163

had been reported to induce intracellular signaling causing the mass secretion of anti-

inflammatory cytokines [13]. The second route states the hemoglobin scavenger receptor

mediates the delivery of hemoglobin to macrophages to fuel the anti-inflammatory effects

within the body because the heme compound within hemoglobin has potent anti-

inflammatory effects [13]. All results obtained for CD163 were statistically significant (all P

values displayed with graphs). An evaluation of CD163 results revealed an unexpected

upregulation of the marker when IL10 was incorporated with THP-1 MΦ, conversely FE

hadn’t caused an upregulation in CD163. It’s clear that FE hasn’t had the desired effect we

predicted, disproving our hypothesis. Across all results (figure 3.3,3.5 and 3.7) it was

revealed that IL-10 was able to induce a higher CD163 expression on cultured THP-1 cells,

however other anti-inflammatory cytokines (IL4) did not increase CD163 expression.


17

This expression pattern implies a functional role of CD163 in the anti-inflammatory response

on monocytes and that IL10 is inducing an M2 phenotype shift. The cause for this

upregulation by IL10 is linked to the marker’s role in blood, as it induces the delivery of

heme to macrophages. Macrophages breakdown heme yielding biliverdin (excreted as

bilirubin), free iron (reabsorbed) and carbon monoxide (CO) [21]. it is the CO molecule which

instigates an anti-inflammatory cytokine secretion and downregulates pro-inflammatory

effects [22]. The role IL10 plays in this process is that it induces the marker to polarize

macrophages to an anti-inflammatory response, suggesting CD163 is indirectly linked to an

anti-inflammatory response. As demonstrated in the graphs above IL-10 is known to

upregulate CD163, it’s likely that IL-10 synthesis will amplify the anti-inflammatory effect.

Investigation into the IL10/CD163 relationship suggest that these two variables shift

phenotype, into M2c macrophages [14]. Other researchers built on this idea and focused on

macrophage polarization induced by intra-plaque hemorrhages, they discovered that

hemoglobin induces a macrophage polarization characterized by expression of IL-10 and

high levels of CD163 [26-27]. It demonstrated that an induction of this type of macrophage

polarization seems to mirror M2c macrophages. Another alternate macrophage differentiation

type is induced by the platelet chemokine CXCL4 [23]. Research into atherosclerosis showed

that CXCL4 neither induced M1 nor M2 polarization in macrophages but induces a different

phenotype with different functional characteristics called M4 macrophage phenotype [24-25].

CXCL4 chemokine has been proven to be able to downregulate CD163 expression during

monocyte-macrophage differentiation, even when strong inducers of CD163 such as IL10 are

present [25]. The results seen in figure 3.7 show that fenugreek dropped CD163 expression,

suggesting that this drop is a result of macrophage shifting from M1 to an M4 phenotype. A

future studies to measure this change using the enzyme: heme oxzgenase-1 and to monitor the

levels of the marker in association with fenugreek extract.


18

4.2 Fenugreek Extract Effect on CD86 Marker

Upon further analysis of figure 3.6 it was noted that both FE concentration 1 & 2 successfully

upregulated the expression of CD86 within monocytes. This expression continued when

THP-1 cells differentiated into M1 macrophages, confirming the shift in phenotype. Figure

3.8 saw an increase in expression when marker was exposed to a higher concentration of FE,

a improvement for the future would be the use of more varied concentrations of FE ranging

from 60ug/ml to 100ug/ml to see if CD86 expression and FE concentration are directly

proportional variables. FE results in figure 3.4 wasn’t statistically significant due to a

preparation error causing contamination within well-plates containing FE meaning that only

one well was successfully detected by flow cytometer. Upon further analysis a particular

sample was found to be consistent and abnormally high; IL4+CD86 test samples across all

experiments where found to have an elevated fluorescence intensity in comparison to FE. The

reason behind this result was intriguing and required further research.

Figure 3.6 and 3.8 above showed CD86 was highly expressed when both concentrations of

FE was applied to cells, however these test samples didn’t display the highest expression.

During monocyte/macrophage activation CD86 becomes rapidly upregulated, a pattern

confirmed by our study but it was interleukin-4 which caused this increase in expression.

IL4+CD86 test sample displayed a higher expression than fenugreek. Interleukin 4 (IL-4) is

produced by alveolar-based basophils in response to an allergic reaction, causing an

upregulation of CD86 production [18], this explains the upregulation of CD86 within our

study. Further research revealed that IL4 is commonly expressed on human monocytic cells,

with the addition of this cytokine to our study a similar overexpression was reported within

THP-1 monocytes. PMA was used to differentiate THP-1 monocytes in this study. Along

with IL4, both were incubated with THP-1 cells for 48 hours. PMA and IL4 combined to

cause an exponential increase in CD86 expression, this was not anticipated [28].


19

Further investigations into the causes of this reaction it was reported by multiple studies that

PMA and IL4 developed a synergistic relationship [28], once PMA was used to differentiate

THP-1 monocytes and incubated alongside IL4 an upregulate of CD86 in the well-plates,

which is why mean fluorescence intensity is elevated in figure 3.6 and figure 3.8. The

pathway needed for IL4 to up-regulate CD86 is the JAK/STAT pathway [18]. This signaling

pathway consists of three main components: a cell surface receptor, janus kinase (JAK) and a

signal transducer and activator of transcription protein (STAT) [29]. To further understand the

role that JAK/STAT pathway plays in this study, future investigations into comparing the

reaction of IL4+CD86 within THP-1 MΦ is needed.

4.3 Limitations of the Study

There where many positive outcomes throughout this study such as the discovery of IL4 and

IL10 role within markers and the significant upregulation of CD86 marker in the presence of

FE. However, there were drawbacks which limited the validity of the study. Due to ethical

constraints connected to animal and human cells an alternate cell line was chosen as the

medium which we tested marker expression. THP-1 cells are a leukemia-derived cell line that

mimic the human monocyte/macrophage-like state. However, there where drawbacks to using

this cell line such as: in vitro differentiation of THP-1 macrophages may cause different

genes to become upregulated during the differentiation process. This cell line has a malignant

background, so it’s difficult to link results to that of normal macrophages [31]. THP-1 cells

where highly sensitive, we experienced a 6-week delay due to cells failing to grow under

cultivated conditions. Cultivation of these cells under specific conditions could result in a

different response to normal somatic cells that would be seen in a natural environment.

Literature searches on THP-1 macrophage interaction revealed that various differentiating

stimuli change the degree of differentiation [30], stimuli like PMA may cause cells to morph

and not resemble tissue macrophages, giving less support to conclusions made between THP-

1 cell line and somatic macrophages. Throughout this experiment only 1 cell line was tested

upon meaning that the results aren’t translatable to other human cells. FE samples caused a

reduction in CD163+IL4 expression, this was thought to be a new M4 phenotype. However,

it’s difficult to measure this and so further investigations into M4 macrophages is needed.

The layout of this experiment had limitations, FE concentrations were added at one-time

point consistently before being incubated for 24 hours, by only adding the extract once to

each well the effect of fenugreek may have stimulated anti-inflammatory effects earlier than

24 hours.


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THP-1 MΦ surface receptors may have released the markers into the plasma during the 24-

hour incubation period and so the marker may have been expressed earlier than flow

cytometry analysis procedure. Given more time an improvement to this study would be the

incorporation of other methodology such as a comparison of genes within THP-1 cells in

order to characterize the monocytic cell line and its mature form. Flavonoids appear to

regulate the expression of many genes, with the use of gene expression it maybe possible to

isolate which genes become upregulated in the presence of flavonoids. This technique could

help reveal information on the molecular mechanism that flavonoids use to produce their

effects. By using microarray assay to compare THP-1 patterns of gene expression, genes

patterns would help describe the distinction between THP-1 monocytes and macrophage.

Gene expression technique could be incorporated to detect which genes induce the

macrophage polarization, which would give further understanding behind the macrophage

plasticity mechanisms.

4.4 Conclusion

The current work carried out confirmed that the fenugreek extract exerted an anti-

inflammatory effect and has shown indicators that it may be able to modulate expression of

M1 and M2 markers in a human macrophage. However, the cytokines IL4 and IL10, which

where initially implemented as positive controls variables produced an unexpected response

in both markers, which hindered the overall effects of FE. The mechanism as to how

fenugreek produced its effects is still a mystery and would require additional work. Positives

outcomes of this study was the identification of a synergistic relationship between cytokines

and markers, these chemical signals work alongside the cells to defend the body against

external threats. More work should be done to investigate further into the cytokine-

macrophage relationship. Future experiments involving fenugreek should be carried out with

the addition of the stimulant at various time points, which would allow for analysis of the

relationship between marker and corresponding receptor. Introducing a greater range of

fenugreek concentration, to see if a lower concentration would have a similar or greater effect

on the marker expression than at 30ug/ml. The exclusion of cytokines from the study would

remove the abnormally high expression of the marker, so fenugreek is being measured

against THP-1 MΦ without the positive controls effecting the overall results and to confirm

the exact effect of fenugreek on macrophage polarization.


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