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 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 non- catalytic 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 TLR-4 TLR-9 IL-6 INOS TNF-a Blocks Inflammatory Response CELL

Model: Enterocyte signaling in the pathogenesis of NEC Lumenalbacteria “Injury” Pathways “Protective” Pathways “Protective” Pathways Normal State Hypoxia, Infection, Prematurity Necrotizing Enterocolitis TLR4-LPS ? TLR9 - CpG-DNA Bacterial DNA Endotoxin

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) Quantity (uL) 10x Running Buffer2.5 5 mmol DNTPS1 50mM MgCl(2)0.75* all by # Primer (gene)1.25of samples H(2)O18.25 Taq0.25 Template (sample)1

SAMPLE GROUPS MediaLPS 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 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 ctrlLPS LPS+CpG CpG IL-6  -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