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American Journal of Immunology 10 (2): 73-87, 2014
ISSN: 1553-619X
©2014 Science Publication
doi:10.3844/ajisp.2014.73.87 Published Online 10 (2) 2014 (http://www.thescipub.com/aji.toc)
Corresponding Author: Radiah C. Minor, Department of Animal Sciences, School of Agriculture and Environmental Sciences,
North Carolina Agricultural and Technical State University, 1601 E. Market St. Greensboro, NC. 27411, USA
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MORINGA TEA BLOCKS ACUTE LUNG
INFLAMMATION INDUCED BY SWINE CONFINEMENT
DUST THROUGH A MECHANISM INVOLVING TNF-α
αα
α
EXPRESSION, C-JUN N-TERMINAL KINASE
ACTIVATION AND NEUTROPHIL REGULATION
1
Mykea Mcknight,
1
Jabria Allen,
1
Jenora T. Waterman,
1
Steven Hurley,
2
Joshua Idassi and
1
Radiah C. Minor
1
Department of Animal Sciences,
2
The Cooperative Extension Program,
School of Agriculture and Environmental Sciences,
North Carolina Agricultural and Technical State University, 1601 E. Market St. Greensboro, NC, 27411, USA
Received 2014-03-21; Revised 2014-04-01; Accepted 2014-05-08
ABSTRACT
Plant based products represent a promising alternative to conventional treatments for inflammation. Moringa
oleifera Lam is a tree rich in proteins, vitamins, minerals and a variety of phytochemcals with health benefits.
Among the reported health benefits are antioxidant and anti-inflammatory properties. The purpose of this study
was to investigate whether tea brewed from dried Moringa leaves would abrogate inflammation in a mouse
model of acute lung inflammation induced by LPS or extracts prepared from dust collected from a swine
confinement facility (DE). Mice were offered water or Moringa tea for seven days. Tea consumption was
significantly greater than that of water consumption on days 1 and 6, but there were no significant differences
in weight gain or food consumption. On day seven, mice from both groups were forced to inhale, via intranasal
challenge, either Phosphate Buffered Saline (PBS), Lipopolysaccharide (LPS) [10 µg mL
−1
] or DE [10%].
Compared to mice that drank water, mice that drank Moringa tea had significantly less protein (p<0.05) and
cellular influx (p<0.0001) into the lung after inhalation of 10% DE. No difference in neutrophil migration into
the lungs of water and M. tea groups after LPS or DE challenge was detected. But mice that drank tea had
significantly (p<0.05) more neutrophils with apoptotic morphology after DE challenge. TNF-α expression 24 h
after inhalation of 10% DE, was significantly higher (p<0.05) in lungs of M. tea mouse group as compared to
water group. This increase in TNF-α was accompanied by higher levels of pro and anti-inflammatory
cytokines. Finally, activation of c-Jun N-terminal Kinase (JNK) in lungs of M. tea+DE group 24 h post
inhalation was decreased. Taken together these results suggest that Moringa oleifera leaf tea exerts anti-
inflammatory properties on acute lung inflammation induced by swine confinement dust through a mechanism
involving neutrophil regulation and JNK activation.
Keywords: Inflammation, Lung, Agricultural Dust, Complimentary and Alternative Medicine
1. INTRODUCTION
Inflammation is a physiological response that protects
a host against external and internal assault. It is the first
response of a host to an infection or irritation, meant to
destroy (or contain) a damaging agent, initiate repair and
restore function of damaged tissue. In this instance,
inflammation is a necessary and helpful process.
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However, inflammation, particularly chronic or
persistent low-grade inflammation can be associated with
deleterious effects, can lead to tissue damage and is
associated with chronic diseases like cancer, diabetes,
heart disease allergies and asthma (Freund et al., 2010).
For decades, plant-based materials have been
considered an important source of natural products used
for health. Natural products continue to be important to
consumers and recently there has been an increase in the
popularity in Complementary and Alternative Medicine
(CAM). Consumers increasingly demand such products
to promote heath and prevent disease. According to a
report by the Centers for Disease Control (CDC), the use
of dietary supplements is common among the U.S. Adult
population and it is estimated that over 50% of
Americans over 20 years old report using supplements
(Bailey et al., 2011).
Moringa oleifera, or Moringa is tree native to the
sub-Himalayan regions of India, Pakistan, Bangladesh
and Afghanistan, that is now widely cultivated and
naturalized in many places around the globe including
the US (Anwar et al., 2007). Moringa has health-
promoting bioactive and nutritive components that
increase its potential as a natural supplement in treating
disease. Among the many positive benefits, it has
immune modulating, antioxidant and anti-inflammatory
properties (Fahey, 2005; Muangnoi et al., 2012). All
parts of the Moringa oleifera tree are edible and
associated with health benefits. Eating the plant leaves,
or consuming leaf-concentrate, or extract can positively
affect health and immunity and immune cells (Fahey,
2005; Anwar et al., 2007). Experiments in guinea pigs
has shown that β-sitosterol isolated from an n-butanol
extract of Moringa oleifera seeds led to the synthesis and
release of Th2 cytokines in a model for allergic asthma
(Mahajan and Mehta, 2011). Methanolic extract of
Moringa oleifera enhanced the phagocytosis of
neutrophils while ethanolic extract (50%) of M. oleifera
leaves promoted phagocytosis by macrophages in the
immunosuppressed mouse model (Sudha et al., 2010;
Gupta et al., 2010). Ethanolic extract of Moringa seeds
in contrast, showed immunosuppressive effect and
caused down-regulation of macrophage phagocytosis of
carbon particles (Mahajan and Mehta, 2010). Taken
together, these data illustrate that different parts of the
Moringa tree exhibit a myriad of immunomodulatory
activities. Much of these modulatory activities require
further exploration. The goal of this study was to
investigate the immune modulating and anti-
inflammatory properties of tea prepared from Moringa
leaves using a model of acute lung inflammation.
2. MATERIALS AND METHODS
2.1. Preparation and Analysis of 1% (w/v)
Moringa Tea
Dried Moringa leaves (from plants grown in
Winston-Salem NC) were used to prepare M. tea. Dried
Moringa leaves (30 g) were steeped in 3 L of boiling
hot distilled deionized water for 30 min. After steeping,
the M. tea was filtered through cheese cloth to remove
large particles, then through a funnel lined with 3 M
filter paper to remove smaller particles. Finally, M. tea
was filter sterilized through 0.22 micron filter and
stored at 4°C until used.
2.2. Preparation of Dust Extracts
Dust extracts were prepared as previously described
(Pender et al., 2014). Briefly, one gram of swine
facility dust was combined with 10 mL of phosphate
buffered saline solution without calcium and
magnesium vortexed for 1 min. The mixture was left to
stand at room temperature for 1 h and then centrifuged
at 948 xg for 10 minutes at room temperature. The
supernatant was transferred to a new tube and
centrifuged again for 10 minutes at the highest speed.
The supernatant was sterilize by filtration (0.22 micron
filter) and stored at in-80°C until used.
2.3. Mice and Dosing
A total of 30 female wild-type Balb/c mice, between
the ages of 7-8 weeks were used. All animals were fed
standard rodent chow (Purina 5001) and provided water
or M. tea ad libitum throughout the study. Fresh water
and M. tea was offered daily for 7 days. At the time of
each water/M. tea change, fluid consumption was
recorded by subtracting the amount of water left from
the amount of water given. The average daily
consumption per mouse was determined by dividing the
total water consumption of each cage was by the
number of mice per cage. In addition, weights and
chow consumption were recorded daily. All mice were
maintained
in the laboratory animal research unit of
North Carolina Agrisultural and Technical State
University and used in accordance with applicable
regulations after
institutional approval.
2.4. Induction of Inflammation
Because mice are obligate nose breathers, liquid is
aspirated during normal respiration and inflammation was
induced via the intranasal challenge method as described
elsewhere (Minor et al., 2012). Briefly, mice were lightly
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anesthetized with isoflurane. Then either
sterile/endotoxin free phosphate-buffered saline (PBS)
(Fisher Scientific, Pittsburgh, Pennsylvania), LPS
[Sigma, St. Louis, MO #L4391 µg mL
−1
], or dust
extracts (10%) was applied with a micropipette (50
µL
−1
) to the nares. After inhalation of the droplet was
complete the mice were returned to their cage.
2.5. Necropsy, Tissue, BAL Collection and
Protein Assay
On day 8, 24 h after intranasal installation, mice
were euthanized by CO
2
inhalation and
Bronchoalveolar Lavage (BAL) was performed on the
left lobe of the lung with 1X PBS. Total protein levels
within the BAL fluid were measured by Bradford
assay (Bio-Rad Laboratories, Hercules, CA) per
manufacturer’s instructions.
2.6. Cell Analysis
For cell analysis BAL was centrifuged at 300×g for
10 min. The cell pellets from bronchoalveolar lavage
fluid were resuspended in 200 µL of PBS. For flow
cytometry analysis side scatter and forward Scatter
analysis of the resuspended cells fluid was conducted
using an Accuri C6 flow cytometer (BD), collecting
25,000 cells. The percent cells in the live gate were
plotted. Differential cell stain was also performed on
resuspended cells as described (Minor et al., 2012).
Briefly, cells were affixed to glass slides using a
Shandon cytospin 4 (Thermo Fisher Scientific Waltham,
Massachusetts) at 700 rpm for 5 min. Slides were dried,
fixed, stained using HEMA-3 stain (Fisher Scientific,
Pittsburg, Pennsylvania) and using a compound light
microscope with a 100× oil immersion lens, a minimum
of 200 total cells per slide were identified and counted
based on color and morphology. Differential cell analysis
to distinguish normal and apoptotic morphology was
conducted on the HEMA-3 stained slides counting a
minimum of 200 neutrophils per slide. Apoptosis was
assessed by changes in nuclear morphology as described
in (Rytila et al., 2006). Briefly normal neutrophils have
nuclei with at least two lobes connected by chromatin
bridges. Cells determined to be undergoing apoptosis had
lost chromatin bridges and had condensed nuclei.
Pictures of cells was completed using a Zeiss Axio
Imager m2m Optical Microscope (Carl Zeiss Microscopy
GmbH, Jena, Germany) at 1000X magnification.
2.7. Cytokine Assays
TNF-α ELISA (Bio Legend, San Diego, California)
was performed on the BAL fluid following
manufacturer’s protocols. Plates were read at 405 nm
using a VersaMax microplate reader (Molecular
Devices, Sunnyvale, CA). Harvested right lung lobes
were stored in-80°C and later used to prepare protein
extracts for cytokine array analysis and western blot.
Cytokine array analysis was performed using the
Proteome Profiler Mouse Cytokine Array Kit, panel A
catalog#ARY006 (R and D systems, Minneapolis,
MN) with extracts prepared from the middle lobes of
the right lung that were homogenized per
manufacturer’s instructions. Briefly, lung tissue was
homogenized with a pellet pestle grinder and cordless
mixer motor in PBS with 1X protease inhibitors
(Pierce Rockford, IL#78439). Triton X-100 was added
to the homogenate to a final concentration of 1%. The
samples were frozen at-80°C, overnight. After
thawing, the samples were centrifuged at 10,000 g for
5 min to remove cellular debris. Protein quantitation
of sample extracts was performed (Pierce Rockford,
IL). Each of the four protein array blots supplied with
the kit were incubated with 300 µg of total protein.
2.8. Western Blot
Protein extraction for western blot were prepared
by homogenizing upper right lung lobes with a pellet
pestle grinder and cordless mixer motor in 200 mL of
1X RIPA buffer supplemented with protease and
phosphatase inhibitors (Pierce Rockford, IL).
Homogenates were centrifuged at 10,000 g for 5 min.
Whole cell lysates were collected and quantified by
protein assay (Pierce Rockford, IL). Total protein
extracts [30 µg well] from lung were separated via
SDS-PAGE, transferred to nitrocellulose membranes
and probed with anti-mouse p-JNK (Santa Cruz, Santa
Cruz California (sc-6254) and anti-mouse JNK (sc-
7345). Cytokine arrays and western blot images were
developed and analyzed for densitometry using the
ChemiDoc system (Bio Rad, Hercules, CA) imaging
system and ImageLab software.
2.9. Statistical Analysis
GraphPad Prism version 5 (La Jolla, CA) was used
to create graphs and conduct all statistical analysis. P
values less than 0.05 were considered significant. There
were five mice in each of the experimental condition;
water+PBS, M. tea+PBS, water+LPS, M. tea+LPS,
water+10% DE, M. tea+DE. Statistical power as
determined using a statistical power calculator from
DSS research was greater than 95% for data where
there were 2 fold increases.
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3. RESULTS
3.1. Dosing and Weight
In order to evaluate the anti-inflammatory role of
Moringa tea in acute lung inflammation induced by
occupation dust exposure we developed a mouse model.
Mice were given fresh Moringa tea prepared from dried
Moringa leaves daily for seven days. We found no
significant differences in weight gain or food consumption
between the two groups; water and M. tea (Fig. 1B and C).
Moreover, mice in the tea group consumed M. tea at the
same rate and at times better (Fig. 1A; days 1 and 6-8) than
the control mice consumed water.
3.2. Analysis of Inflammation; Cellular Influx
and Protein Expression
Inflammatory responses are associated with increases
in vascular permeabilization and cellular influx. Figure
2A shows that while the level of total protein increased
after exposure to LPS and 10% DE, mice that consumed
M. tea had significantly less protein in the BAL fluid 24 h
after inhaling the DE as compared to mice that consumed
water (Fig. 2A). To compare cellular influx, flow
cytometry was performed on the BAL fluid collected 24
h after intranasal inhalation of PBS, LPS or 10% DE.
Figure 2B and C show that mice that drank water had
significantly more cells in the BAL of mice exposed to
10% DE (Fig. 2B and C). But, cellular influx into the
lung after challenge with DE was significantly (p<0.0001)
lower in mice that consumed Moringa tea (Fig. 2B and
C). These data suggesting that there was less inflammation
in the lung of mice that consumed the M. tea.
In humans and animals, lung challenge with
Lipopolysaccharide (LPS) causes a neutrophil-rich
inflammatory responses and intranasal instillation of
swine confinement dust extracts in mice has been shown
to lead to increased cellular inflammation that is
predominated by neutrophils (Sandström et al., 1994)
(Jagielo et al., 1996; Poole et al., 2009). Differential cell
analysis of the cells collected from the BAL show that
neutrophils were the predominant cell type detected in
the in the lungs 24 h after LPS and 10% DE challenge
(Fig. 2D). Interestingly, there were no significant
differences between the total macrophage, neutrophil, or
lymphocytes (no other WBC types were observed) in the
BAL of mice that consumed water or Moringa tea (Fig.
2D). Through further analysis of neutrophils, using
nuclear morphology to distiguish neutrophil viability we
determined that after LPS challenge in both water and M.
tea groups there were few neutrophils with apoptotic
nuclear morphology (Fig. 3A and B). However, after
challenge with 10% DE there were significantly (p<0.05)
more apoptotic neutrophils (50.3%) present in the mice
that drank the Moringa tea as compared to the mice that
drank water (24.1%) (Fig. 3A and B). Taken together
this suggests that consumption of Moringa Tea resulted
in had less inflammation through a mechanism that
involved neutrophil viability.
3.3. Cytokine Expression
TNF-α, is a cytokine associated with pro-
inflammatory responses. However, divergent roles for
TNF-α have been reported. Van Den Berg et al. (2001)
reported neutrophils exposed to low concentrations [<1.0
ng mL
−1
] of TNF-α have increased survival while higher
concentrations of TNF-α [10-100 ng mL
−1
], induce
apoptosis (Van Den Berg et al., 2001). TNF-α ELISA
was performed on BAL collected 24 h post inhalation.
We report that, as one might expect, TNF-α expression
was induced by LPS and 10% DE both the water and M.
tea groups. However, TNF-α expression was
significantly greater in mice from the M. tea group after
challenge with 10% DE compared to mice in the water
group with the same challenge (Fig. 4). This level of
TNF-α [average 1.0 ng mL
−1
] while not at the level
described by Van Den Berg et al. (2001) to cause
apoptosis it is potentially greater than the level that
provides protection and therefore may have led to the
increased incidence of apoptosis in neutrophils detected
in the BALF collected from mice that consumed M. tea
after inhalation of 10% DE observed in Fig. 3.
In addition to preventing or inducing apoptosis, TNF-
α expression can also induce the expression of other
cytokines that have pro and anti-inflammatory functions.
Therefore we sought to determine the expression levels
of other pro and anti-inflammatory cytokines in the lung
after challenge with 10% DE. Lung protein extracts were
analyzed by cytokine array comparing PBS and 10% DE
challenged mice, since that was the condition that
resulted in a greater significant difference in TNF-α
production. We had positive reactions with 17 of the 40
total cytokine antibodies spotted on the array (Fig. 5A)
and report changes of two-fold or greater in 11 of the 17
cytokines detected. Namely; Tumor Necrosis Factor
(TNF)-α, Keratinocyte Chemoattractant (KC),
Triggering Receptor Expressed on Myeloid cells-1
(TREM-1), Macrophage-Colony Stimulating Factor (M-
CSF), Macrophage Inflammatory Protein-(MIP)1-α,
MIP-2, IL-16, IL-1α/β, Regulated upon Activation,
Normal T cell Expressed and Secreted (RANTES) and
IL-l receptor antagonist (IL-1ra) (Fig. 5B).
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(A)
(B)
(C)
Fig. 1. Fluid, food consumption and weight gain of mice. For (A and B) water and feed consumption was measured daily during the eight
day trial.
Total average water and feed consumption was calculated to find average daily consumption per mouse by dividing the
total amount consumed by the number of mice per cage. Data are expressed as means ±SD.
For water and tea consumption in (A) a
two-way repeated measures ANOVA with a 95% confidence level was used to determine significance (** = p<0.001, * = p<0.05).
(C) All mice were weighed daily on an Arbor 1605 electronic balance.
In all graphs, data are an average of 15 mice per group.
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(A)
(B)
(C)
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(D)
Fig. 2. (A) Protein levels within the BALF were measured 24 h after inhalation challenge. Shown are the average levels for
each condition (n = 5).
One-way ANOVA analysis with Bonferroni’s multiple comparison test with a 95% confidence
level was used to determine significance. (B) Representative dot plots of BALF cells analyzed by flow cytometry for
size and granularity.
(C) Histogram of the flow cytometry data showing the average (n = 5) percent cell data obtained
for each experimental treatment.
One-way ANOVA with Bonferroni’s Multiple Comparison Test with a 95%
confidence level was used to determine significance. (D) For differential cell analysis, data are averaged from an n of
five in each group and are expressed as means ± SD
(A)
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(B)
Fig. 3. (A) Slides prepared for differential cell analysis (Figure 2D) were also used to evaluate neutrophil apoptosis.
A total of
200 neutrophils per slide were counted using 100 X magnifications and oil immersion. Before counting, slides were de-
identified.
Data are an average (n = 5) in each condition and are expressed as means ± SD. One-way ANOVA with
Bonferroni’s Multiple Comparison Test with a 95% confidence level was used to determine
significance. (B)
Representative slides showing normal (black arrows) and apoptotic neutrophils (red arrow heads)
Fig. 4. TNF-α ELISA on BAL fluid collected 24h post inhalation challenge. ELISA was conducted per manufacturer’s directions.
Data are an average of n = 5 in each group. Data are expressed as means ± SD and were compared using a two-tailed unpaired
Student t test with a 95% confidence level
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(A)
(B)
Fig. 5. (A) Lung tissue lysates were prepared by homogenization in 1X ice cold PBS with protease inhibitors. Each array was probed
with 300 µg of lung lysate prepared from pooled lung tissues (n = 5 for each blot).
(B) Densitometry was performed on the
spots with gel-doc system software. First density for the pair of spots were averaged, then averages were then normalized to
the average density of the reference spots.
Next, the normalized densities of lung lysates from PBS treated mice were
subtracted from the normalized densities of the DE treated lungs (M. tea+DE-M. tea+PBS and water+DE-water+PBS).
Finally the normalized M.
Tea+DE with PBS subtracted values were divided by water + DE with PBS subtracted values.
The
dotted line represents the 2 fold cut-off
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(A) (B)
Fig. 6. (A) Total protein extracts from lungs were separated via SDS-PAGE, transferred to nitrocellulose membranes
and probed for
p-JNK and total JNK. (A) Each lane represents extracts prepared from an individual animal (n = 5).
(B) Average change in
pJNK activation was determined through densitometric analysis of the protein bands for JNK and pJNK.
Data is expressed as
a ratio between the density of p-JNK divided by that of total JNK (n = 5). Data are expressed as means ± SD and were
compared using a two-tailed unpaired Student t test with a 95% confidence level
3.4. MAP Kinase Activation
The c-Jun N-terminal Kinases/Stress-Activated
Protein Kinase (JNK/SAPK) is a member of the
Mitogen-Activated Protein Kinase (MAPK)
superfamily, which are activated in response to LPS
and contribute to inflammatory responses (Ip and
Davis, 1998). Inhibition of JNK/MAPK signaling is a
targeted strategy of reducing inflammation
(Kaminska, 2005). Therefore, we investigated whether
tea brewed from dried Moringa leaves would inhibit
the activation of JNK/MAPK pathway. Western blot
analysis of protein extracts prepared from the lungs 24
h post intranasal challenge show decreased activation
of JNK in animals that drank Moringa tea as
compared to those that drank water (Fig. 6).
4. DISCUSSION
Asthma and allergy combined are the leading chronic
diseases in the United States and respiratory disease is
one of the main chronic conditions among farmers and
their families (Freund et al., 2010). Farm environments
contain several particulates (e.g., feed grain, fecal matter,
animal dander and traces of bacteria and mold) that may
pose inhalation exposure risk to agricultural workers and
their families. Inhalation of endotoxins and carbon
dioxide at levels above the recommended health
threshold limits (Mc Donnell et al., 2008) can lead to
acute and chronic airway inflammation (Schierl et al.,
2007). Poole et al. (2009) reported increased cellular
inflammation, predominated by neutrophils, in mouse
BALF following a single intranasal instillation of DE.
Vitamins, minerals, antioxidants and other active
compounds present in plants and plant-based materials
can modulate immune responses and alleviate
inflammation. Interestingly, four out of the five classes of
drugs that are presently being used as an asthma treatment
originate from herbs (Ziment and Tashkin, 2000). For
example, green tea brewed from the leaves of the plant
Camellia sinensis was found to block the expression of an
anti-inflammatory cytokine (Li et al., 2007) and extracts
prepared from grape seeds which have potent antioxidant
and anti-inflammatory effects were shown to alleviate
inflammation and asthma associated pathologies in a mouse
model of ovalumin-induced asthma (Mahmoud, 2012).
Additionally supplementation of mouse diets with vitamin
D [1,25 hydroxyvitamin D], one of the vitamins found in
Moringa (Anwar et al., 2007), reduced signatures of lung
inflammation (i.e., lung neutrophilia and BAL levels of IL-
8) in mice exposed to organic dust daily for two weeks
compared to mice fed a standard chow (naturally containing
low levels of vitamin D) (Golden et al., 2013).
We designed a study to investigate the potential of
tea prepared from dried Moringa leaves to block acute
lung inflammation after exposure to LPS or dust
collected from a swine confinement facility. We found
that mice that consumed Moringa tea had fewer cells
within the BAL fluid in the lung 24 h after inhaling 10%
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DE as detected by flow cytometry (Fig. 2) despite having
significantly higher levels of TNF-α than controls in
response to the DE (Fig. 4). This is consistent with a
previous report by (Mahajan et al., 2009) using a model
of ovalbumin-induced airway-inflammation that showed
extracts from Moringa seeds improved total differential
cell counts in blood and bronchoalveolar lavage fluid,
but not levels of TNF-α (Mahajan et al., 2009). More
recently, a study by (Kooltheat et al., 2014) was
published evaluating the effect of an ethyl acetate
fraction of Moringa (MOEF) prepared from fresh leaves
on cytokine production by human macrophages exposed
to cigarette smoke extract. It was reported that
pretreatment of human monocyte derived macrophages
with varying concentrations of MOEF abrogated TNF-α,
IL-6 and IL-8 cytokine production to LPS exposure
(Kooltheat et al., 2014). This further illustrates an anti-
inflammatory role for Moringa. While TNF-α, is a
cytokine typically associated with pro-inflammatory
responses, divergent roles for TNF-α have been reported
whereby there are instances where it also has
immunosuppressive function. Studies have shown TNF-
α can both provoke and protect against the induction of
apoptosis in neutrophils. Van den Berg et al. (2001)
reported neutrophils exposed to low concentrations [<1.0
ng ml
−1
] of TNF-α have increased survival while higher
concentrations of TNF-α [10-100 ng ml
−1
], induce
apoptosis (Van Den Berg et al., 2001). Here we show
data consistent with this phenomenon, whereby greater
numbers of neutrophils with apoptotic morphology were
detected in the experimental condition that led to the
highest level of TNF-α expression (M. tea+DE) (Fig. 3
and 4) suggesting that the level of TNF-α within the
BAL [average 1.0 ng mL
−1
] of mice that consumed
Moringa tea and inhaled DE may have led to
increased incidence of apoptosis of neutrophils in the
lung. The decrease in cellular influx detected by flow
cytometry but not by differential cell staining may
have been due to the dead cells being gated out during
cell acquisition as apoptotic cells do not display
similar forward and side scatter as viable neutrophils.
Reports on Moringa’s effect on inflammatory cells
such as neutrophils has been inconsistent. Where some
report with animal models that Moringa consumption
leads to increases in neutrophils and neutrophil
function; others find that Moringa can cause decreases in
neutrophil levels within the blood (Gupta et al., 2010;
Owolabi et al., 2012; Isitua and Ibeh, 2013). Further
studies to elucidate the effect of Moringa on neutrophils,
specifically induction of apoptosis are needed.
In addition to abrogating or inducing apoptosis, TNF-
α expression can also induce the expression of other
cytokines that have anti-inflammatory functions. Other
pro-and anti-inflammatory cytokines were found in this
study to be differentially expressed by mice that
consumed Moringa tea. We report that after DE
exposure, mice that consumed M. tea had 2 fold or
higher increases in the levels of pro-
inflammatory/chemotactic cytokines (IL-1β and α, KC,
M-CSF, RANTES, MIP-2 and MIP1-α, in the lung, as
compared to mice that drank water. We also observed
increases in immunosuppressive cytokines IL-6, IL-1Ra
and TREM-1 (De Bie et al., 2002; Okada et al., 1995;
Little and Cruikshank, 2004; Gibot and Massin, 2006;
Giamarellos-Bourboulis et al., 2008).
IL-1β or TNF-α expression can lead to increased IL-16
expression by epithelial cells (Little et al., 2003) a cytokine
with an immunomodulatory role in asthmatic inflammation
(De Bie et al., 2002; McFadden et al., 2007). IL-1Ra is an
antagonist to IL-1α/β signaling in the lung (Wilmott et al.,
1998). It is produced at high levels by neutrophils in
response to LPS stimulation or exposure to TNF-α
(McColl et al., 1992; Nguyen et al., 2010) and in a guinea
pig model of late asthmatic reactions (Okada et al., 1995).
Ning and colleagues investigated mice with acute lung
inflammation and reported a positive correlation in
expression pattern between TREM-1 and TNF-α whereby
both increased with LPS treatment (Liu et al., 2010).
TREM-1 is expressed by neutrophils, monocytes and
macrophages (Bouchon et al., 2000). Moreover, the
active form of vitamin D, 1, 25 (OH) (2) D (3), found in
high levels in Moringa (Anwar et al., 2007) induces the
expression of TREM-1 by Normal Human Bronchial
Epithelial (NHBE) cells. Activation of TREM-1 leads to
expression of β-defensin-2 and TNF-α (Rigo et al.,
2012). During bacterial infections TREM-1 accelerates
the elimination of bacteria and therefore has a protective
role in innate immune responses, but is associated with
overwhelming inflammation (Bouchon et al., 2001;
Lagler et al., 2009). The membrane form of TREM-1 can
be cleaved into a soluble form, sTREM that is released
into microenvironment (Gomez-Pina et al., 2007). While,
high levels of sTREM-1 have been found in patients with
severe forms of allergic asthma (Bucova et al., 2012), it has
been suggested that sTREM-1 may have an anti-
inflammatory role, by acting through a mechanism whereby
sTREM blocks interactions of membrane-bound TREM-1
with its natural ligand in a way similar to the recognized
interaction between the soluble form of the TNF-α receptor
and membrane TNF-α receptor (Gibot and Massin, 2006;
Mykea Mcknight et al. / American Journal of Immunology 10 (2): 73-87, 2014
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Giamarellos-Bourboulis et al., 2008). Furthermore, it has
been suggested that sTREM could be used as therapeutic
for inflammatory conditions such as rheumatoid arthritis
and sepsis (Kim et al., 2012; Wang et al., 2012). The
cytokine array used in this study measures both
membrane and soluble forms of TREM-1, therefore,
further analysis of the effect of Moringa on this cytokine
is needed. In summary, cytokine analysis suggest the
within the BAL there was increased expression of anti-
inflammatory cytokines (IL-16, IL-Ra) that may have
helped to mitigate inflammation in the lung.
The Mitogen Activated Protein Kinase proteins
(MAPKs) are a group a intracellular signal transduction
enzymes found in yeasts, animals and plant cells
(Ichimura, 2002). They are triggered in response to
extracellular signals and instruct new gene expression, cell
survival, growth/proliferation, differentiation or death
(Kim and Choi, 2010) in response to the stimuli. LPS
treatment of human neutrophils has been shown to activate
JNK and JNK activation was shown to be unimportant for
LPS-induced TNF-α expression (Arndt et al., 2004). This
is constant with our findings where JNK activation is
lowest in the condition that gave the highest expression of
TNF-α (M. tea+DE). Arndt et al. (2004), demonstrated
that systemic treatment of mice with JNK inhibitor
SP600125 resulted in inhibition of JNK activation,
decreased neutrophil recruitment and a decrease in the
microvascular leak in the lungs after LPS inhalation
(Arndt et al., 2005). M. oleifera pod, root, leaf and fruit
extracts were reported to block inflammatory responses
of a macrophage cell line stimulated with LPS by
inhibiting MAPK, NF-κB activation activation
(Muangnoi et al., 2012; Lee et al., 2013). In keeping
with these studies, we report here that activation of JNK
was abrogated in mice that consumed tea made from the
leaves of Moringa oleifera and that this was associated
with decreases in BAL protein levels (a measure of lung
leakage) (Fig. 2A) and decrease in the levels of viable
neutrophils (Fig. 3) within the lungs after inhalation of
swine confinement facility dust. The effect that Moringa
may have had on other MAPK pathway proteins was not
evaluated here but is of interest. Research into the
immune modulating aspects of Moringa is ongoing and
follow up studies, investigating the role of Moringa on
cell (neutrophil) migration and viability are planned.
5. CONCLUSION
Moringa is being cultivated and sold as a nutritional
supplement. It has been reported to alleviate a host of
conditions including hepatoxicity (Hamza, 2007),
neuropathic pain (Khongrum et al., 2012), oxidative
damage (Kirisattayakul et al., 2012) and inflammation
(Muangnoi et al., 2012; Lee et al., 2013). Here we
present data that suggests immunoprotective and anti-
inflammatory properties for Moringa that involves
regulation of neutrophils in airway inflammation.
6. ACKNOWLEDGEMENT
The researchers thank Mr. Livingston Mawutor of
Winston-Salem, NC for growing and providing the
Moringa leaves used in this study. We also acknowledge,
Chakia McKlendon and Dawn Conklin for technical
assistance and Dr. Sergey Yarmolenko for technical
assistance with neutrophil images. This work supported
in part by Grant numbers; NCX-270-5-12-120-1 (to
R.C.M), NCX-255-5-11-120-1 (to J.T.W.) and 2011-
38821-30967 (to J.T.W.) each from the USDA National
Institute of Food and Agriculture. Its contents are solely
the responsibility of the authors and do not necessarily
represent the official views of the National Institute of
Food and Agriculture.
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