THE ROLE OF TLR-4 IN THE ROLE OF TLR-4 IN INTESTINAL HEALINGINTESTINAL HEALING

Nectrotizing Enterocolitis (NEC)Nectrotizing Enterocolitis (NEC)

Most common and most Most common and most lethal disease affecting lethal disease affecting the GI tract of the the GI tract of the premature infant.premature infant.0.9 to 2.4 per 1000 live 0.9 to 2.4 per 1000 live births; 2% of all births; 2% of all neonatal deaths neonatal deaths Mortality 40%-90%Mortality 40%-90%Risk factors: Risk factors: prematurity (90% prematurity (90% between 30-32 weeks), between 30-32 weeks), respiratory respiratory insufficiency, hereditary insufficiency, hereditary heart diseaseheart disease

INFLAMMATORY RESPONSEINFLAMMATORY RESPONSE

Inflammation - biological Inflammation - biological response of vascular tissues to response of vascular tissues to harmful stimuli - pathogens, harmful stimuli - pathogens, damaged cells, or irritantsdamaged cells, or irritantsA protective attempt by the A protective attempt by the organism to remove the organism to remove the injurious stimuli as well as injurious stimuli as well as initiate the healing process for initiate the healing process for the tissue – NOT synonymous the tissue – NOT synonymous with infectionwith infectionAbsence of inflammation - Absence of inflammation - wounds and infections never wounds and infections never heal and progressive heal and progressive destruction of the tissue destruction of the tissue compromises the survivalcompromises the survivalUnchecked inflammation can Unchecked inflammation can also lead to a host of diseases, also lead to a host of diseases, such as hay fever, such as hay fever, atherosclerosis, and atherosclerosis, and rheumatoid arthritisrheumatoid arthritis

TOLL-LIKE RECEPTORSTOLL-LIKE RECEPTORSToll-like receptors - class of single membrane-spanning non-Toll-like receptors - class of single membrane-spanning non-catalytic receptors that recognize molecules derived from catalytic receptors that recognize molecules derived from microbes once they have breached physical barriers and activated microbes once they have breached physical barriers and activated immune cell responses immune cell responses Believed to play a key role in the innate immune systemBelieved to play a key role in the innate immune systemTLRs are a type of TLRs are a type of pattern recognition receptorpattern recognition receptor - recognize - recognize molecules that are broadly shared by pathogens but molecules that are broadly shared by pathogens but distinguishable from host moleculesdistinguishable from host moleculesPresent in both vertebrates and invertebratesPresent in both vertebrates and invertebratesTLR – 4 is believed to promote the release of signaling proteins TLR – 4 is believed to promote the release of signaling proteins which spark inflammatory response, activated by LPSwhich spark inflammatory response, activated by LPSTLR-9 believed to be the mediator of the inflammatory response, TLR-9 believed to be the mediator of the inflammatory response, activated by CpGactivated by CpGCpG – DNA sequence, cytosine and guanine separated by CpG – DNA sequence, cytosine and guanine separated by phosphate, links the two nucleosides togetherphosphate, links the two nucleosides togetherLPS - LPS -

IEC-6IEC-6

TLR-4 – TLR-9TLR-4 – TLR-9

LPS CpG

TLR-4 TLR-9

IL-6

INOS

TNF-a

Blocks

Inflammatory Response

CELL

Model: Enterocyte signaling in the pathogenesis of NECModel: Enterocyte signaling in the pathogenesis of NEC

Lumenal Lumenal bacteriabacteria

““Injury” PathwaysInjury” Pathways “ “Protective” PathwaysProtective” Pathways

Normal StateNormal State

Hypoxia, Infection, PrematurityHypoxia, Infection, Prematurity

Necrotizing EnterocolitisNecrotizing Enterocolitis

TLR4-LPSTLR4-LPS ??TLR9 - CpG-DNATLR9 - CpG-DNA

Bacterial DNABacterial DNA EndotoxinEndotoxin

PREVIOUS STUDYPREVIOUS STUDY

Whether LPS treatment affects TLR-9 – CpG Whether LPS treatment affects TLR-9 – CpG receptorreceptorTested in IEC-6 cells, epithelial rat cells, Tested in IEC-6 cells, epithelial rat cells, participate in inflammatory responseparticipate in inflammatory responseIndependent variable – LPS concentrationIndependent variable – LPS concentrationDependent variable – presence of activated Dependent variable – presence of activated TLR-9TLR-9TLR-9 expression measured via Western blot TLR-9 expression measured via Western blot – presence of signaling proteins– presence of signaling proteinsResult: The expression of TLR-9 in IEC-6 Result: The expression of TLR-9 in IEC-6 cells is unchanged with LPS treatmentcells is unchanged with LPS treatment

PURPOSEPURPOSE

To determine the effect of CpG on the To determine the effect of CpG on the production of signaling proteins which production of signaling proteins which activate the inflammatory responseactivate the inflammatory response

HYPOTHESISHYPOTHESIS

The addition of CpG will activate TLR-9 The addition of CpG will activate TLR-9

which in turn will inhibit the productionwhich in turn will inhibit the production

of signaling proteins that activate theof signaling proteins that activate the

inflammatory response.inflammatory response.

MATERIALSMATERIALS

Reaction MixReaction Mix

Sterile pipetsSterile pipets

Western blot machineWestern blot machine

PCR machinePCR machine

SamplesSamples

Deionized waterDeionized water

Agarose GelAgarose Gel

Gel coloringGel coloring

Marking dye (for Marking dye (for samples)samples)

VortexVortex

Basic laboratory safety Basic laboratory safety equipmentequipment

Crystal violet dyeCrystal violet dye

Sterile tubesSterile tubes

PCR running bufferPCR running buffer

REACTION MIX (RECIPE)REACTION MIX (RECIPE)

Quantity (uL)Quantity (uL)

10x Running Buffer10x Running Buffer 2.52.5

5 mmol DNTPS5 mmol DNTPS 11

50mM MgCl(2)50mM MgCl(2) 0.750.75 * all by #* all by #

Primer (gene)Primer (gene) 1.251.25 of of samplessamples

H(2)OH(2)O 18.2518.25

TaqTaq 0.250.25

Template (sample)Template (sample) 11

SAMPLE GROUPSSAMPLE GROUPS

MediaMediaLPSLPS

LPS + CpGLPS + CpGIFNIFN

IFN + CpGIFN + CpGTNF-aTNF-a

TNF-a + CpGTNF-a + CpGIL-1IL-1

IL-1 + CpGIL-1 + CpGCytomixCytomix

Cytomix + CpGCytomix + CpGCpGCpG

NTC - nothingNTC - nothing

PROCEDURE (PT. 1)PROCEDURE (PT. 1)

*note* ALL work was done on ice*note* ALL work was done on ice2 quantities of reaction mix created, 1 for each gene 2 quantities of reaction mix created, 1 for each gene tested, IL-6, TNF-a, INOS, b-actin – marked tested, IL-6, TNF-a, INOS, b-actin – marked accordinglyaccordingly1uL sample pipetted into respective tubes + 24uL 1uL sample pipetted into respective tubes + 24uL reaction mixreaction mix1 extra tube per gene – contained just reaction mix, 1 extra tube per gene – contained just reaction mix, control for cross-contaminationcontrol for cross-contaminationLiquid on all walls tapped downLiquid on all walls tapped downPCR (polymerase chain reaction) machine warmed PCR (polymerase chain reaction) machine warmed up while samples preparedup while samples preparedAll tubes All tubes sterilely sterilely cappedcappedTubes placed into PCR machine to run overnight Tubes placed into PCR machine to run overnight (standard run time ~ 6 hours)(standard run time ~ 6 hours)

PROCEDURE (PT. 2)PROCEDURE (PT. 2)

*note* All work again done on ice*note* All work again done on iceTest tubes removed from PCR machineTest tubes removed from PCR machineAgarose gel created w/ designated number of wells, Agarose gel created w/ designated number of wells, 4uL running dye added, poured into mold to cool4uL running dye added, poured into mold to cool6uL coloring added to each sample6uL coloring added to each sampleGel removed from mold, placed into gel machine, Gel removed from mold, placed into gel machine, immersed in running bufferimmersed in running buffer5uL running ladder pipetted into first well5uL running ladder pipetted into first well6uL running dye added to all samples, 20uL of each 6uL running dye added to all samples, 20uL of each sample pipetted into respective wellssample pipetted into respective wellsGel “run” at 200-300 volts for ~ 30 minutesGel “run” at 200-300 volts for ~ 30 minutesGel removed when samples traveled far enough, Gel removed when samples traveled far enough, placed under UV light, photographedplaced under UV light, photographed

RESULTSRESULTSINOS

B-actin

RESULTSRESULTS

dfd

B-actin

RESULTSRESULTS

ctrlctrl LPSLPSLPS+LPS+CpGCpG CpGCpG

IL-6IL-6

-Actin-Actin

B- actin = control – establishes that the amount of sample pipetted into each well was the same

IL-6 = one of the signaling genes

CONCLUSIONCONCLUSION

Insufficient evidence to prove/disprove Insufficient evidence to prove/disprove that addition of CpG affects the that addition of CpG affects the production of INOS, TNF-aproduction of INOS, TNF-a

IL-6 shows a decrease in activation IL-6 shows a decrease in activation with the presence of CpGwith the presence of CpG

EXTENSIONSEXTENSIONS

Greater sample size, more than 2 tubes Greater sample size, more than 2 tubes dedicated to a particular genededicated to a particular gene

Wider range of tests for gene Wider range of tests for gene expression, more genesexpression, more genes

Another variable other than CpG, Another variable other than CpG, different types of cellsdifferent types of cells