Transcript Slide 1

Leptin regulation of ocular immunity
Emma Jakes
Authentic Science Research Program
Manchester-Essex Regional High School, Manchester-by-the-Sea, MA 01944
Abstract
Leptin regulation of ocular immunity
Emma Jakes
Manchester-Essex Regional High School, Manchester-by-the-Sea, MA
Teacher Dr. Maria Lonnett Burgess, Manchester-Essex High School
Mentor, Dr. Andrew Taylor, Boston University School of Medicine
Boston University, Boston, MA
The immune system is comprised of many molecules that
regulate the ability of the body to fight infection. In the eye,
the concept of “immune privilege” is defined as the lack of
harmful inflammatory response due to the composition of
fluids that are present. The eye is vulnerable to a variety of
internal and external pathogens that can threaten survival,
especially in humans. To avoid this, evolution has provided a
spectrum of defense mechanisms that can neutralize
pathogens invading the eye’s fluid compartments.
Tissue Culture Media
• DMEM plus 10% Fetal Bovine Serum (FBS)
• Phenol red-free DMEM plus 10% FBS
• Serum-free RPMI1640 Phenol red-free Media (SFM)
• Phenol red-free RPMI1640 plus 10% FBS
Another hormone, a-Melanocyte Stimulating (a-MSH) is also
suspected to confer this privilege. The goal of this project
was to determine if Leptin would promote inflammation in
the eye by regulating nitric oxide, or NO. Varying
concentrations of Leptin were tested for their ability to affect
NO on certain cell lines. Each experiment yielded relatively
similar results, showing that different concentrations of
Leptin did not affect NO release from the cells. Therefore,
our original hypothesis was not confirmed by these
experiments.
Objectives
To research Leptin’s role in immune regulation by
determining if:
• Leptin acts as a pro-inflammatory hormone on
macrophages in vitro.
• Elicited increased levels of NO in macrophages are
suppressed by a-MSH.
• Leptin acts as
antagonist to
anti-inflammatory
factors.
Conclusions
• Leptin does not enhance NO levels produced by RAW and
J774 cells in vitro .
• a-MSH appears to suppress NO at concentrations low as
0.3ng/ml
• Experiments using different concentrations of Leptin were
expected to increase NO levels.
• Experiments using different concentrations of a-MSH were
expected to suppress NO levels by suppressing
inflammation.
• NO levels that registered in the assay were between 0 and
400mM in Fig 2, but the sodium nitrate stock-based curve for
this experiment was not constant.
• Neither Leptin or a-MSH enhanced or suppressed NO levels
respectively
• Reasoning:
• NO reaction happens faster than can be tested
• Cells were old and differentiation could have
changed their properties
• Leptin may react differently in-vitro
Fig 3. NO levels suppressed by concentrations of a-MSH.
Procedure
• Culture J774.2 and RAW cell line (DMEM)
• Centrifuge to solid pellet for counting
• Pipette cell concentration into 96 well plates, including
duplicates, and incubate
• Wash cells with new media (SFM)
• Prepare concentrations of needed solutes, pipette and
successively dilute concentrations, incubate for 24 hours
1
Leptin, a hormone that regulates metabolism and other
essential body functions, is thought to be involved in
systemic inflammatory response. The effect of Leptin on the
other regulating factors responsible for ocular immune
privilege is described in this study.
Results
Methods
A
B
2
3
4
5
6
CTRLA
CTRLB
a-MSH
a-MSH
a-MSH
a-MSH
LPS
SFM
LPS
LPS
LPS
LPS
SFM
No LPS
SFM
SFM
SFM
SFM
a-MSH
a-MSH
a-MSH
a-MSH
LPS
SFM
LPS
LPS
LPS
LPS
SFM
No LPS
SFM
SFM
SFM
SFM
Table 1. Dilutions used to test NO levels of LPS stimulated cells.
Dilutions used were: a-MSH: 5mg/ml → 1/100 → 50ug/ml → 1/100 →
500ng/ml → 1/42 → 12ng/ml → ⅓ →4ng/ml → 1/3 → 1.2ng/ml →
1/3 → 0.4ng/ml; LPS: 10mg/ml → 1/100 → 100ug/ml → 1/25 →
4ug/ml
400
350
300
aMSH 3 ng/ml
aMSH 1 ng/ml
aMSH 0.3 ng/ml
aMSH 0.1 ng/ml
LPS
No LPS
250
200
150
100
50
References
0
NO Levels (uM)
16
12
Leptin 3 ng/ml
Leptin 1 ng/ml
Leptin 0.3 ng/ml
0.1 ng/ml
LPS
No LPS
10
8
4
2
0
NO Levels (uM)
Fig 5. NO levels enhanced by concentrations of Leptin and suppressed
simultaneously by single concentration of a-MSH (little variation in
results, even with the addition of a-MSH).
14
Leptin 3 ng/ml
Leptin 1 ng/ml
Leptin 0.3 ng/ml
Leptin 0.1 ng/ml
aMSH
LPS
No LPS
12
10
8
6
4
2
0
NO Levels (uM)
Fig 2. Cell media being be transferred to a 96-well plate using a
multi-pipette.
RESEARCH POSTER PRESENTATION DESIGN © 2011
www.PosterPresentations.com
2
Lam QL, Lu L. Role of leptin in immunity. Cell Mol Immunol.
2007;4(1):1 13.107-16.
3
Lau CH, Taylor AW. The immune privileged retina mediates an
alternative activation of J774A.1 cells. Ocul Immunol Inflamm.
2009;17(6):380-9.
4
Otero M, Lago R, Gomez R, Dieguez C, Lago F, Gómez-Reino J,
Gualillo O. Towards a pro-inflammatory and immunomodulatory
emerging role of leptin. Rheumatology (Oxford). 2006;45(8):944-50.
5
Taylor AW. Ocular immune privilege. Eye (Lond). 2009;23(10):1885-9.
6
Taylor AW, Kitaichi N, Biros D. Melanocortin 5 receptor and ocular
immunity. Cell Mol Biol (Noisy-le-grand). 2006;52(2):53-9.
7
Taylor AW, Kitaichi N. The diminishment of experimental autoimmune
encephalomyelitis (EAE) by neuropeptide alpha-melanocyte
stimulating hormone (a-MSH) therapy. Brain Behav Immun.
2008;22(5):639-46.
8
http://us.123rf.com/400wm/400/400/phakimata/phakimata0806/phaki
mata080600061/3131934-blue-multi-channel-pipet-used-for-pipettinga-96-well-plate-with-pink-solution-on-white.jpg
14
16
Fig 1. Diagram
of the eye and its
major functions.
Brzoska T, Böhm M, Lügering A, Loser K, Luger TA. Terminal Signal:
Anti-Inflammatory Effects of α-Melanocyte-Stimulating Hormone
Related Peptides Beyond the Pharmacophore. Adv Exp Med Biol.
2010;681:107-16.
Fig 4. NO levels of cells enhanced by Leptin (little variation in results
at lower levels compared to Fig 3.)
6
• Pipette water into new well plate
• Mix sodium nitrite stock for standard curve, add to top
wells
• Mix N-(1-naphthyl)ethylenediamine dihydrochloride
(Component A) with Sulfanilic acid (Component B) to create
Griess Reagent, add to all wells
• Pipette samples into new well plate
• Run NO assay on plate and analyze results
1
9 www.streilein-foundation.org
Acknowledgement
Thank you to Dr. Andrew Taylor and David Yee for mentoring me in
my research, the BU School of Medicine for allowing me the
opportunity to work in their labs and Dr. Burgess for her support
and enthusiasm.
Contact [email protected]
for further information.