dissertation 1

Arrandhev Singh Kambo st20034270
Fenugreek extract in the regulation of the inflammatory
response and polarization in human macrophages
By Arrandhev Singh Kambo ST20034270
Supervised by Dr. Keith Morris

1
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.

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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: ……………………....

3
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.

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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.

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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.
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
1 2 3
4 5 6

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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.

8
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.
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
1 2
3 4
5 6
1 2 3
4 5 6
7 8 9
10 11 12

9
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.
Figure 3.1: Effect of detachment techniquesTrypsin, 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.
0
1000
2000
3000
4000
5000
6000
7000
MeanFluoresceneIntensity/FL4A

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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.
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.
0
5000
10000
15000
20000
25000
30000
MeanFluoresceneIntensity/FL4A

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3.2 Effect of organic fenugreek extract (FE) on CD163 Marker in THP-1 cell
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.
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.
*
*
*
0.00
1000.00
2000.00
3000.00
4000.00
5000.00
6000.00
Isotype CD163+IL4 CD163+IL10 FE
MeanFLuorescenceIntensity/FL4A

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3.3 Effect of organic fenugreek extract (FE) on CD86 marker in THP-1 cell
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.
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.
*
*
0
5,000
10,000
15,000
20,000
25,000
Isotype CD86 + IL4 CD86 + IL10 FE
MeanFluoroscenceIntensity/FL2A

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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.
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.
*
*
*
* *
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
Cells Isotype CD163 CD163+IL-4 CD163+IL10CD163+FE 1CD163+FE 2
MeanFLuorescenceIntensity/FL4A

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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.
*
*
*
*
*
0.0
2000.0
4000.0
6000.0
8000.0
10000.0
12000.0
cells only isotype cd86 cd86 + IL4 CD86 +IL10 CD86 + FE
conc 1
CD86 + FE
conc 2
MeanFluorescenceIntensity/FL2A

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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.
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.
*
*
*
*
0.00
500.00
1000.00
1500.00
2000.00
2500.00
3000.00
Cells Isotype CD163+IL4 CD163+IL10 CD163+FE 1 CD163+FE 2
*
*
*
*
*
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
isotype cd86 cd86 +IL4 cd86 +IL10 CD86+FE 1 CD86+FE 2

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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.

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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.

19
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].

20
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.

21
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.

22
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