Crystal violet, APOBrDU, and Cytochrome C assay results showed that L. major inhibits apoptosis in R

1
The Effect of Leishmania major Infection on
Macrophage Apoptosis Katy Kemnetz, Caitlin Mahan,
Eamon Maloney, Emily Swanson, Jonathan
Weyerbacher, Britta Zoeller Center for Tropical
Disease Research and Training, University of
Notre Dame
Abstract
Experimental Flow Chart
Western Blot
APO-BrDU
6A
Leishmania major is an intracellular parasite
that is transmitted through the bite of the
female phlebotomine sand fly. It invades the
macrophages of its host, where it survives by
modulating the host immune response in favor of
parasite development. This infection leads to
cutaneous leishmaniasis, a self-healing form of
the disease. The focus of our research is to
determine, using the macrophage cell line RAW
264.7, how L. major inhibits macrophage apoptosis
as a mechanism for increasing its own survival.
Infected macrophages treated with cycloheximide
(CHX), a pro-apoptotic agent, were found to
maintain higher levels of growth than untreated
infected macrophages. This increase in cell
growth was caused by a decrease in CHX-induced
apoptosis. Cytochrome C levels were found to be
higher in the cytoplasmic fraction of uninfected
CHX-treated macrophages than in the infected
CHX-treated macrophages, suggesting an
intracellular mechanism for apoptosis. Levels of
Bcl-xL, an anti-apoptotic protein, were found to
be lower in uninfected CHX-treated macrophages,
further demonstrating the mechanism through which
L. major suppresses apoptosis in RAW 264.7
macrophages.
Crystal Violet Cell growth assay to infer cell
death
4A
4B
31 7
Med EtOH CHX Med EtOH CHX
std
APO-BrDU/FLOW Cytometry Assay to determine if
cells are undergoing apoptosis
uninfected
infected
6B
APO-BrDU staining
APO-BrDU staining
Cytochrome C Assay Assay to determine if
cytochrome c is released into the cytosol
act std Med EtOH CHX Med EtOH CHX
std act
DNA content
DNA content
uninfected
infected
6C
4D
4C
Western Blot Western blot of anti-apoptotic
Bcl-xL protein to determine levels of expression
31 7
Introduction
Protozoan parasites of the genus Leishmania
infect 2 million people every year, with 350
million others at risk. The various members of
the genus can produce symptoms ranging from a
cutaneous form, marked by skin lesions, to a
visceral form marked by spleen and liver
swelling, ending ultimately in death. L. major,
the parasite used during the course of this
study, produces a single boil on the skin which
spontaneously heals within five to six months.
The disease is transmitted when Leishmania
promastigotes, found in the saliva of the
sandfly, are deposited into the hosts tissue as
the fly feeds. Once inside the tissue, the
parasites are ingested by macrophages where they
transform into the amastigote form and begin to
multiply. Studies have shown that one effect of
Leishmania infection could be the suppression of
apoptosis in the hosts cells. Apoptosis, or
programmed cell death, is one possible way in
which cells can defend themselves against
parasite infection. In the following study, we
examine the effect of L. major infection on
apoptosis in the murine macrophage cell line, RAW
264.7.
Med EtOH CHX Med EtOH CHX
std
APO-BrDU staining
uninfected
infected
Crystal Violet
APO-BrDU staining
6D
DNA content
DNA content
act std Med EtOH CHX Med EtOH CHX std
act

uninfected
infected
pFigure 4 APO-BrDU histograms. The histograms
presented here are as follows (4A) Experiment 2,
uninfected media. (4B) Experiment 2, uninfected
EtOH. (4C) Experiment 1, uninfected CHX. (4D)
Experiment 2, infected CHX.
Figure 6 Western blot. L. major infection
increases expression of Bcl-xL. 30ug of protein
were loaded and gel was transferred onto
nitrocellulose membrane for analysis. Conditions
were stdstandard, actactin standard, medmedia,
EtOH, and CHX, both infected and uninfected. The
blots are as follows (6A) Experiment 1, exposure
of 1 minute. (6B) Experiment 1, actin control.
(6C) Experiment 2, exposure of 1 minute. (6D)
Experiment 2, actin control. The UC condition
shows less protein expression as compared to the
IC condition.
absorbance

Figure 1 Apoptotic pathway diagram. This
segment of the apoptosis pathway details the part
of the pathway with which this study is
concerned. Diagram courtesy of Upstate Group, Inc.
Conclusions
Cytochrome C Assay

• Crystal violet, APO-BrDU, and Cytochrome C assay
  results showed that L. major inhibits apoptosis
  in RAW 264.7.
• The Cytochrome C assay and Bcl-xL assay showed
  that L. major affects the apoptotic pathway above
  Bcl-xL.
• This, along with other literature, suggests that
  the suppression of apoptosis is due to a
  characteristic of the parasite, not a
  characteristic of the macrophage cell line. It
  also shows that this characteristic of the
  parasite is something that affects the apoptotic
  pathway above Bcl-xL.
• Future Experiments
• Future experiments will need to assay expression
  of other proteins in the intracellular apoptotic
  pathway, such as pro-apoptotic proteins Bax and
  Bad, in order to discover the mechanism by which
  L. major suppresses apoptosis. To determine if
  the parasites interaction with macrophage
  receptors has an effect on apoptosis, known
  receptors used by Leishmania can be studied using
  knockout mutants. Cells can also be infected with
  dead parasites to confirm if the cause is related
  to a characteristic of the parasite rather than
  its activity. For example, it has been suggested
  that it is the LPG coat of the parasite that
  causes apoptosis suppression and not any activity
  of the parasite itself. Further experiments could
  also performed to confirm whether it is this
  characteristic of Leishmania parasites that
  causes apoptotic suppression.

Uninfected
pellet
supernatant
CHX EtOH med CHX EtOH
med std
Figure 2 Crystal Violet. L. major infection of
CHX-treated cells increases cell growth. Controls
were media and EtOH carrier control. Cells
infected with L. major and uninfected cells were
incubated with 5ug/ul CHX for 12 hours. There is
a statistically significant (pby student t-test) increase in the amount of cell
growth after CHX treatment when the cells are
infected as opposed to uninfected.
5A
31.3
Materials and Methods
5B
act std med EtOH CHX med
EtOH CHX std
Cell line Used RAW 264.7, a murine macrophage
cell line grown in RPMI (10 FBS, 1
Penicillin/Streptomycin, 1 l-Glutamine). Parasite
s Line Used Leishmania major (V1), Friedlin
strain grown in Medium 199 10 heat-inactivated
FBS, 100 ug/ml penicillin, 100 ug/ml
streptomycin, 2mM L-glutamine, 40mM HEPES, 0.1mM
adenine (in 50mM HEPES), 5 mg/ml hemin (in 50
triethanolamine) and 1 mg/ml biotin. Metacyclic
promastigotes for infecting macrophages were
isolated through the use of a ficoll gradient.
Macrophages were plated, infected with
promastigotes at a ratio of 201, and harvested
12 hours later. A small portion of the cells for
each experiment were set aside to determine
infection rate. Cell condition/experiment
Uninfected cells media, ethanol carrier and 5
ug/ml CHX Infected cells media, ethanol carrier,
and 5 ug/ml CHX. Crystal Violet Assay Cells for
each condition were harvested and fixed with 1
glutaraldehyde. Cells were labeled with 0.1
Crystal Violet dye, and 0.2 Triton X was used
to permeabilize cell membranes. The absorbance of
the crystal violet dye was then measured at a
wavelength of 590 nm. APO-BrDU Assay Two
replicates of each cell condition were used in
this assay as well as positive and negative
controls for the APO-BrDU kit. Cell fixation,
permeabilization and staining were performed by
following the protocol from the APO-BrDU kit (BD
Pharmigen). Cells for each condition and its
replicate were then analyzed with FLOW cytometer
(Coulter, Epics XL). Cytochrome C Assay
Subcellular fractionation using Buffer A (Young
et al, 1997), an isotonic buffer, was performed
to separate protein of the cytosol from protein
of the cell organelles for each cell condition
into separate Eppendorf tubes. A Western Blot was
then run for protein found in the cytosol. Actin
control (Sigma) was also used to ensure that
equal amounts of protein were in each lane.
Primary antibody anti-Cytochrome C (BD
Pharmigen). Secondary antibody anti-mouse.
Primary actin antibody anti-actin (Ab-1) mouse
mAb (JLA20) (CalBioChem). Secondary actin
antibody Goat anti-mouse IgM (CalBioChem).
Western Blot 30 ug of protein in sample buffer
for each cell condition were boiled, loaded, and
run on SDS-PAGE gel. Actin control (Sigma) was
used again to ensure equivalent amounts of
protein were found in each lane. Primary
antibody anti-Bcl-XL(Cell Signaling Technology,
2762). Secondary antibody Goat
anti-rabbit-horseradish peroxide (BD Pharmigen).
Primary actin control antibody anti-actin (Ab-1)
mouse mAb (JLA20) (CalBioChem). Secondary actin
antibody Goat anti-mouse IgM (CalBioChem).
APO-BrDU
pellet
supernatant
Infected
pellet
supernatant
CHX EtOH med CHX EtOH med
std
40.4 31.3
5C
Acknowledgements
5D
We would like to thank the following people for
their help and contribution to this project Dr.
Mary Ann McDowell McDowell Lab Michael
Donovan Kristen Leary Lori Clark Dr. Michelle
Whaley Dr. Martin Tenniswood Tenniswood Lab Soma
Roy Welsh Lab Judy Narvaez Ferdig Lab
act std med EtOH CHX med EtOH
CHX std
supernatant
pellet
Figure 5 Cytochrome C assay. L. major infection
decreases Cytochrome C in supernatant. Conditions
were stdstandard, medmedia, EtOH, and CHX, both
infected and uninfected for all conditions. The
pellet and supernatant fractions of these
conditions are indicated. There is a decrease in
Cytochrome C for the CHX supernatant condition in
the infected cells as opposed to the uninfected
cells. This figure is representative of two
experiments. The blots are as follows (5A)
Experiment 1, uninfected, exposure of 25 minutes.
(5B) Experiment 1, uninfected, actin control.
(5C) Experiment 1, infected, exposure of 25
minutes. (5D) Experiment 1, infected, actin
control.
Figure 3 APO-BrDU. L. major infection decreases
apoptosis. Conditions were uninfected media,
uninfected EtOH, uninfected CHX, infected media,
infected EtOH, and infected CHX. Cells were
prepped with an APO-BrDU kit and counted by FLOW
cytommetry. The APO-BrDU run showed a 76
decrease in apoptosis for the infected CHX
compared to the uninfected CHX condition.
For more information on the Effect of L. major on
Macrophage Apoptosis, visit www.nd.edu/bzoeller