Inflammation 2013

Sample & Assay Technologies

InflammaTools: Complete research tools to study Inflammation regulation

Raed Samara, Ph.D.R&D Scientist

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Introduction: Inflammation

Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues.

Microbes/InfectionTissue Damage

Acute Inflammation

Chronic InflammationInfection ClearanceTissue Homeostasis

Pre-cancer & CancerChronic Inflammatory Diseases

Cytokines & ChemokinesSignaling PathwaysImmune system composition

EpigeneticChanges

mRNAChanges

Inflammation experimental design & QIAGEN

Gene Expression� RT-PCR

Epigenetics� miRNA� DNA methylation� Histone modifications

Functional Studies� Reporter assays� siRNA/shRNA

Protein Expression� Cytokine levels

Basic principles of qRT-PCR: Overview

Real-Time PCR

Amplify and simultaneously quantify target DNA

Reverse Transcription Real-Time PCR

Amplify and simultaneously quantify mRNA

For more information and webinars on real-time PCR, visit:

www.sabiosciences.com/seminarlist.php

Experimental overview: Gene expression analysis

Stimulate CellsRT-PCR Arrays or Assays

Data Analysis

Isolate RNA

Isolate DNA

Gene expression in Inflammation

Infection ClearanceTissue Homeostasis

EpigeneticChanges

mRNAChanges

Acute Inflammation

Chronic Inflammation

Inflammation studies: Gene expression

The transcription factor cMyb is upregulated in naïve CD4 T cells.

The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb on naïve CD4 T cell maturation (to Th1, Th2, or Th3)?

Experiment: Knockdown cMyb in human peripheral CD4+ T cells. Examine gene changes using the Human Th1-Th2-Th3 RT2 Profiler PCR Array (PAHS-034).

Results:

Conclusions: Knockdown of cMyb in human peripheral CD4+ T cells decreased the expression of Th2 cytokine genes, and negatively affected Th2 cell maturation in primary human peripheral blood T cells.

Gene expression in Inflammation

EpigeneticChanges

mRNAChanges

Acute Inflammation

Inflammation studies: Gene expressionLimited material

Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of inflammation.

Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of inflammation. What roles do TLRs play in MDF?

Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and normal colon. Convert mRNA to cDNA. Pre-amplify cDNA with the Mouse TLR PreAMP primer mixes (PBM-0018). Analyze differential gene expression with the Mouse TLR RT2

Profiler PCR Arrays (PAMM-018)

Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and normal colon. Pre-amplify cDNA with the Mouse TLR PreAMP primer mixes (PBM-0018). Analyze differential gene expression with the Mouse TLR RT2 Profiler PCR Arrays (PAMM-018)

Results:

Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and normal colon. Pre-amplify cDNA with the Mouse TLR PreAMP primer mixes (PBM-0018). Analyze differential gene expression with the Mouse TLR RT2 Profiler PCR Arrays (PAMM-018)

Results:

Conclusion: MDF induces TLR2 gene expression in MDF compared to normal colon, suggesting a link between TLR2 and MDF.

Epigenetics: Overview

Structural Gene NFκB BS

Activated Transcription Factors

p53 BS

Protein “A”

mRNA ”A”

MeMeMe

Histones

Histone-DNA Interactions

DNA Methylation

Me AcMe Me MeMe

DNA Methylation

miRNAshRNAsiRNA

Transcription Initiation Complex

For more information and webinars on Epigenetics, v isit:www.sabiosciences.com/seminarlist.php

miRNA reagents from QIAGEN

3’ UTR Reporters

Target Identification Expression

Isolation

Function

miRNA mimics & inhibitors

miScript miRNA PCR Arrays

miRNeasy

miRNAStudies

miRNA in Inflammation

EpigeneticChanges(miRNA)

mRNAChanges

Acute Inflammation

Inflammation studies: miRNA function

Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1) are a class of players that mediate this resolution.

FASEB J. 25, 1–17 (2011)

Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1) are a class of players that mediate this resolution. Can RvD1-mediated resolution happen through miRNAs?

FASEB J. 25, 1–17 (2011)

Experiment: Treat mice with zymosan A to induce acute inflammation (murine peritonitis), in the presence or absence of RvD1, for 4, 12, or 24 hours (representing onset, maximum, and resolution phases). Collect RNA from leukocytes. Analyze miRNA expression with the Mouse miRNome miRNA PCR Array (MIMM-216Z)

FASEB J. 25, 1–17 (2011)

Results:

Conclusion: RvD1 regulated resolution by controlling the expression of miR-219 and miR-146b. These miRNAs are the first identified miRNAs in resolvin resolution circuits.

p53 BS

Protein “A”

mRNA ”A”

MeMeMe

Histones

DNA Methylation

Me AcMe Me MeMe

DNA Methylation

miRNAshRNAsiRNA

DNA methylation in Inflammation

EpigeneticChanges

(DNA Methylation)

mRNAChanges

Acute Inflammation

Inflammation studies: DNA methylation

ANCA Vasculitis is characterized by microvascular inflammation caused by activated neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are silenced in normal individuals.

ANCA Vasculitis is characterized by microvascular inflammation caused by activated neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are silenced in normal individuals.Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing?

Experiment: Isolate gDNA from neutrophils and treat with methylation-sensitive and methylation-dependent restriction enzymes (EpiTect Methyl DNA Restriction Kit 335451). Analyze DNA methylation by real-time PCR using primers specific for CpG islands in PR3 (MePH23428-1A) and MPO (MePH22382-1A).

Results:

Conclusion: Methylation of CpG islands is a mechanism that controls the expression of MPO, but not the expression of PR3.

The EpiTect Methyl System uses the MethylScreen™ Technology provided under license from Orion Genomics, LLC.

p53 BS

Protein “A”

mRNA ”A”

MeMeMe

Histones

DNA Methylation

Me AcMe Me MeMe

DNA Methylation

miRNAshRNAsiRNA

Histone modification in Inflammation

EpigeneticChanges

(Histone Modifications)

mRNAChanges

Acute Inflammation

Inflammation studies: Histone modification

Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays an important role in T cell differentiation in the periphery.

Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays an important role in T cell differentiation in the periphery. Does TCF-1 control the differentiation of Th17 cells, and how?

Experiment: Isolate naïve T cells from spleens of mice. Induce Th17 cell differentiation. Collect chromatin and process with the EpiTect ChIP one-day kit (334471) for chromatin immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR.

Experiment: Isolate naïve T cells from spleens of mice. Induce aTh17 cell differentiation. Collect chromatin and process with the EpiTect ChIP one-day kit for chromatin immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR.

Results:

Open chromatin = increased expression

Experiment: Isolate naïve T cells from spleens of mice. Induce Th17 cell differentiation. Collect chromatin and process with the EpiTect ChIP one-day kit (334471) for chromatin immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR.

Results:

Conclusion: TCF-1 mediates the repression of IL-17 locus during T cell development by chromatin modifications.

Reporter Construct GFP/firefly luciferaseTATAbox

Tandem repeats of TRE

Transcriptional Regulatory Elements (TRE), which es tablish thespecificity of each reporter

Cignal Reporter Assays: Complete SolutionReporter assays: Overview

TF EGFP FL

Upstream SignalingEvents

Reporter assays for Inflammation

EpigeneticChanges

mRNAChanges

Acute Inflammation

Inflammation studies: Reporter assay

Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory activity of CPS is largely unknown.

Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory activity of CPS is largely unknown. What signaling pathways are induced upon CPS recognition?

Experiment: Reverse-transfect cells with 10 dual-luciferase reporter assays, individually, on a reporter array (Cignal Finder 10-pathway Reporter Array; CCA-108L). Induce transfected cells with CPS, and measure luciferase levels to determine which pathway(s) is(are) activated by CPS

Results:

Conclusions: NFkB is the major signaling pathway activated in response to CPS.

shRNA/siRNA function

p53 BS

Protein “A”

mRNA ”A”

MeMeMe

Histones

DNA Methylation

Me AcMe Me MeMe

DNA Methylation

miRNAshRNAsiRNA

Gene knockdown for Inflammation

EpigeneticChanges

mRNAChanges

Acute Inflammation

Inflammation studies: siRNA knockdown

Resident alveolar macrophages (AM) constitute the first line of defense against invading lung Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience increased mortality and bacterial outgrowth and dissemination.

The Journal of Immunology, 2008, 180: 3594–3600.

Resident alveolar macrophages (AM) constitute the first line of defense against invading lung Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience increased mortality and bacterial outgrowth and dissemination. What is the mechanism by which morphine does this?

Experiment: MH-S cells (murine AM cells) were transfected with either a negative control SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2 were measured after treating transfected cells with morphine and Streptococcus pneumoniae

Experiment: MH-S cells (murine AM cells) were transfected with either a negative control SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2 were measured after treating transfected cells with morphine and Streptococcus pneumoniae.

Results:

Experiment: MH-S cells (murine AM cells) were transfected with either a negative control SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2 were measured after treating transfected cells with morphine and Streptococcus pneumoniae.

Results:

Conclusion: Morphine reduces levels of Streptococcus pneumoniae -induced MIP-2 from AM cells in a TLR9-dependent manner.

Chemokine expression in Inflammation

EpigeneticChanges

mRNAChanges

Acute Inflammation

Inflammation studies: Chemokine levels

Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined.

The Journal of Immunology, 2010, 184: 6275–6282

Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. Could this be due to defective chemokine secretion leading to delayed priming of adaptive immunity?

Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine levels using the Multi-Analyte ELISArray Kit.

Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine levels using the ELISArray.

Results:

Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine levels using the Multi-Analyte ELISArray Kit.

Results:

Conclusion: Reduced levels of leukocyte chemoattractant factors including CCL2 and CCL5 at early timepoints post-infection could explain why diabetic mice are more prone to TB.

Conclusions

QIAGEN offers many methods to study molecular and cellular mechanisms involved in Inflammation:

� Gene Expression� RT2 Profiler PCR Arrays & Assays� RT2 PreAMP Primer Mixes

� Epigenetics� miScript miRNA PCR System� EpiTect Methyl qPCR Arrays� EpiTect ChIP qPCR Arrays

� Functional Studies� Cignal Reporter Assays� SureSilencing shRNA Plasmid

� Protein expression� ELISArray

www.sabiosciences.com

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Inflammation 2013

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