Download THE ROLE OF TLR-4 IN INTESTINAL HEALING Nectrotizing

THE ROLE OF TLR-4 IN
INTESTINAL HEALING
Nectrotizing Enterocolitis (NEC)
Most common and most
lethal disease affecting
the GI tract of the
premature infant.
0.9 to 2.4 per 1000 live
births; 2% of all
neonatal deaths
Mortality 40%-90%
Risk factors:
prematurity (90%
between 30-32 weeks),
respiratory
insufficiency, hereditary
heart disease
INFLAMMATORY RESPONSE
Inflammation - biological
response of vascular tissues to
harmful stimuli - pathogens,
damaged cells, or irritants
A protective attempt by the
organism to remove the
injurious stimuli as well as
initiate the healing process for
the tissue – NOT synonymous
with infection
Absence of inflammation wounds and infections never
heal and progressive
destruction of the tissue
compromises the survival
Unchecked inflammation can
also lead to a host of diseases,
such as hay fever,
atherosclerosis, and
rheumatoid arthritis
TOLL-LIKE RECEPTORS
Toll-like receptors - class of single membrane-spanning noncatalytic receptors that recognize molecules derived from
microbes once they have breached physical barriers and activated
immune cell responses
Believed to play a key role in the innate immune system
TLRs are a type of pattern recognition receptor - recognize
molecules that are broadly shared by pathogens but
distinguishable from host molecules
Present in both vertebrates and invertebrates
TLR – 4 is believed to promote the release of signaling proteins
which spark inflammatory response, activated by LPS
TLR-9 believed to be the mediator of the inflammatory response,
activated by CpG
CpG – DNA sequence, cytosine and guanine separated by
phosphate, links the two nucleosides together
LPS -
IEC-6
TLR-4 – TLR-9
LPS
CpG
CELL
TLR-4
TLR-9
IL-6
INOS
Blocks
TNF-a
Inflammatory Response
Model: Enterocyte signaling in the pathogenesis of NEC
Bacterial DNA
Lumenal
bacteria
Endotoxin
Hypoxia, Infection, Prematurity
“Injury” Pathways
“Protective” Pathways
?
TLR4-LPS
TLR9 - CpG-DNA
Necrotizing
Normal
Enterocolitis
State
PREVIOUS STUDY
Whether LPS treatment affects TLR-9 – CpG
receptor
Tested in IEC-6 cells, epithelial rat cells,
participate in inflammatory response
Independent variable – LPS concentration
Dependent variable – presence of activated
TLR-9
TLR-9 expression measured via Western blot
– presence of signaling proteins
Result: The expression of TLR-9 in IEC-6
cells is unchanged with LPS treatment
PURPOSE
To determine the effect of CpG on the
production of signaling proteins which
activate the inflammatory response
HYPOTHESIS
The addition of CpG will activate TLR-9
which in turn will inhibit the production
of signaling proteins that activate the
inflammatory response.
MATERIALS
Reaction Mix
Sterile pipets
Western blot machine
PCR machine
Samples
Deionized water
Agarose Gel
Gel coloring
Marking dye (for
samples)
Vortex
Basic laboratory safety
equipment
Crystal violet dye
Sterile tubes
PCR running buffer
REACTION MIX (RECIPE)
10x Running Buffer
5 mmol DNTPS
50mM MgCl(2)
Primer (gene)
H(2)O
Taq
Template (sample)
Quantity (uL)
2.5
1
0.75
1.25
18.25
0.25
1
* all by #
of samples
SAMPLE GROUPS
Media
LPS
LPS + CpG
IFN
IFN + CpG
TNF-a
TNF-a + CpG
IL-1
IL-1 + CpG
Cytomix
Cytomix + CpG
CpG
NTC - nothing
PROCEDURE (PT. 1)
*note* ALL work was done on ice
2 quantities of reaction mix created, 1 for each gene
tested, IL-6, TNF-a, INOS, b-actin – marked
accordingly
1uL sample pipetted into respective tubes + 24uL
reaction mix
1 extra tube per gene – contained just reaction mix,
control for cross-contamination
Liquid on all walls tapped down
PCR (polymerase chain reaction) machine warmed
up while samples prepared
All tubes sterilely capped
Tubes placed into PCR machine to run overnight
(standard run time ~ 6 hours)
PROCEDURE (PT. 2)
*note* All work again done on ice
Test tubes removed from PCR machine
Agarose gel created w/ designated number of wells,
4uL running dye added, poured into mold to cool
6uL coloring added to each sample
Gel removed from mold, placed into gel machine,
immersed in running buffer
5uL running ladder pipetted into first well
6uL running dye added to all samples, 20uL of each
sample pipetted into respective wells
Gel “run” at 200-300 volts for ~ 30 minutes
Gel removed when samples traveled far enough,
placed under UV light, photographed
RESULTS
INOS
B-actin
RESULTS
dfd
B-actin
RESULTS
ctrl
LPS
LPS+
CpG
CpG
IL-6
b-Actin
B- actin = control – establishes that the amount of
sample pipetted into each well was the same
IL-6 = one of the signaling genes
CONCLUSION
Insufficient evidence to prove/disprove
that addition of CpG affects the
production of INOS, TNF-a
IL-6 shows a decrease in activation
with the presence of CpG
EXTENSIONS
Greater sample size, more than 2 tubes
dedicated to a particular gene
Wider range of tests for gene
expression, more genes
Another variable other than CpG,
different types of cells