tlr and inflammation 2013
DESCRIPTION
TRANSCRIPT
Sample & Assay Technologies
Toll-like Receptors in InflammationRaed Samara, Ph.D.R&D Scientist
Sample & Assay Technologies QIAGEN webinars
Access recorded webinars and slides
Title, Location, Date 2
http://sabiosciences.com/home.php
Sample & Assay Technologies QIAGEN webinars
Access recorded webinars and slides
Title, Location, Date 3
Sample & Assay Technologies Keep up to date: Follow Pathway focused biology on Facebook
Latest information on pathway and disease research, resources and demos.
Sample & Assay Technologies Biology-Focused Solutions for Pathway Analysis
SABiosciences is now a Company
Title, Location, Date 5
http://sabiosciences.com/home.php
Sample & Assay Technologies Introduction: Inflammation
Title, Location, Date 6
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
mRNAChangesTLR
TLR
Sample & Assay Technologies Toll-like Receptors (TLR)
Rakoff-Nahoum & Medzhitov. 2009. Nat Rev Cancer
TLR8
TLR10
Recognize molecular patterns derived from:- Microbes- Endogenous molecules
Either:- Directly- Indirectly (with the help of accessory molecules)
Both at:- Plasma membrane- Intracellular compartments
Signal through:- MYD88- TRIF
TLR activation leads to:- Regulation of innate immune response- Regulation of adaptive immune responses- Inflammation- Tissue repair
Sample & Assay Technologies Ligands of TLR
Title, Location, Date 8
Chen et al. 2007. Int’l Immunopharm
Sample & Assay Technologies Expression of TLR
TLR are expressed in a wide variety of cells
Title, Location, Date 9
Ospelt & Gay. 2010. Int’l J Biochem Cell Biol
Sample & Assay Technologies TLR signaling
Title, Location, Date 10
Foster & Medzhitov. 2009. Clin Imm
TAK1
Sample & Assay Technologies TLR signaling
Title, Location, Date 11
Uematsu & Akira. 2007. JBC
Sample & Assay Technologies TLR signaling
Induction of 3 classes of transcription factors
Title, Location, Date 12
Medzhitov & Horng. 2009. Nat Rev Imm
Post-translational activationof transcription factors after TLR stimulation
Transcription factors are induced during the primary response
Transcription factors are cell-specific
Sample & Assay Technologies TLR in inflammation
TLR activation leads to a complex module-specific inflammatory transcriptional response
Title, Location, Date 13
Medzhitov & Horng. 2009. Nat Rev Imm
Sample & Assay Technologies TLR in inflammation
TLRs form a bridge between innate and adaptive immune responses
Title, Location, Date 14
Parker, Prince, Sabroe. 2007. Clin Exp Imm
Sample & Assay Technologies TLR in inflammation
Title, Location, Date 15
Mills. 2011. Nat Rev Imm
TLR
DC T cells
Sample & Assay Technologies
Title, Location, Date 16
Sample & Assay Technologies Expression of TLR
Expression of TLR and MYD88 in the CNS of EAE-diseased mice
Title, Location, Date 17
EAE is an animal model of multiple sclerosis
Detection of mRNA expression by RT-PCR
Sample & Assay Technologies Effects of modulating TLR activity on EAE
TLR signaling is critical for the induction of EAE and Th1 responses
Title, Location, Date 18
Induction of EAE by active immunization
Knockout of MYD88 protected mice from EAE
Knockout of MYD88 decreased Th1 responses after induction of EAE
Sample & Assay Technologies Which cell type is it?
Title, Location, Date 19
Mills. 2011. Nat Rev Imm
TLR
DC T cells
Sample & Assay Technologies Effects of modulating TLR activity on EAE
Title, Location, Date 20
Transfer reactive T cells (responsible for inducing EAE) in to mice of different strains
Knockout of TLR9 or MYD88 in non-T cells in the host decreased severity of EAE
Sample & Assay Technologies TLR in inflammation
Title, Location, Date 21
Mills. 2011. Nat Rev Imm
TLR
DC T cells
Sample & Assay Technologies
Title, Location, Date 22
Sample & Assay Technologies Effects of TLR activation on Treg cell activity
TLR activation in Tregs suppresses their suppressive functions
Title, Location, Date 23
Sample & Assay Technologies Effects of TLR activation on Treg cell activity
The TLR8-Myd88-IRAK4 pathway is required to reverse the suppressive functions of Tregs
Title, Location, Date 24
Knockout indicated components in Tregs, sort Tregs, co-culture sorted Tregs with naïve CD4+ cells
Sample & Assay Technologies Regulation of TLR signaling
Title, Location, Date 25
Foster & Medzhitov. 2009. Clin Imm
Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation
RT2 Profiler PCR Arrays & Assays
Title, Location, Date 26
http://sabiosciences.com/PCRArrayPlate.php
Profile 84 different genes on one array
Appropriate controls for data normalization, sample quality, and reaction performance
Focus on your questions and papers
Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation
RT2 Profiler PCR Arrays & Assays
Title, Location, Date 27
Array Species
TLR Signaling Pathway Human/Mouse/Rat/Rhesus
NFkB Signaling Pathway Human/Mouse/Rat
NFkB Signaling Targets Human/Mouse/Rat
MAPK Signaling Pathway Human/Mouse/Rat
PI3K-Akt Signaling Pathway Human/Mouse/Rat
TH1 & Th2 responses Human/Mouse/Rat/Rhesus
Interferons & Receptors Human/Mouse/Rat
Others (>140) Varies
Array Species
Antibacterial responses Human/Mouse/Rat
Antiviral responses Human/Mouse/Rat
Antifungal responses Human/Mouse/Rat
Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation
RT2 Profiler PCR Arrays & Assays
Title, Location, Date 28
SampleIsolation & Preparation
Assay Set-up & Analysis
Customer support group:1-888-503-3187
Sample & Assay Technologies Regulation of TLR signaling
Title, Location, Date 29
Quinn & O’Neil. 2011. Int’l Imm
Sample & Assay Technologies miRNA in TLR & inflammation
Title, Location, Date 30
Sample & Assay Technologies miRNA webinars
Title, Location, Date 31
http://sabiosciences.com/home.php
Sample & Assay Technologies miRNA webinars
Title, Location, Date 32
Sample & Assay Technologies
Title, Location, Date 33
Sample & Assay Technologies Expression of miR-181 in hematopoietic lineages
Northern analyses
Title, Location, Date 34
Chen, …, Bartel et al. 2004. Science
Sample & Assay Technologies Expression of miR-181 in hematopoietic lineages
Ectopic expression
Title, Location, Date 35
Chen, …, Bartel et al. 2004. Science
Transduce bone marrow cells with Vector or miR-181; reconstitute mice with transduced BM cells; detect cell lineages by flow
Monocytes Neutrophils nonmyeloid
Sample & Assay Technologies
Title, Location, Date 36
Sample & Assay Technologies Expression of miRNAs after TLR activation
Microarray analyses
Title, Location, Date 37
Taganov, …, Baltimore et al. 2006. PNAS
Induce THP-1 cells (human acute monocytic leukemia) with LPS
Microarray results are validated by RT-PCR
Sample & Assay Technologies Expression of miR-146a is NFkB-dependent
Title, Location, Date 38
Taganov, …, Baltimore et al. 2006. PNAS
Mutations in NFkB binding site in the genomic loci of miR-146a
Sample & Assay Technologies miR-146 targets mediators downstream of TLR
Title, Location, Date 39
Taganov, …, Baltimore et al. 2006. PNAS
Transfect cells with TRAF6 or IRAK1 reporter constructs, transfect cells with miRNAs, read luciferase activity
Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation
miScript miRNA PCR Arrays & Assays
Title, Location, Date 40
http://sabiosciences.com/mirna.php
Profile 84 different miRNAs on one array
Appropriate controls for data normalization, sample quality, and reaction performance
Focus on your questions and papers
Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation
miScript miRNA PCR Arrays & Assays
Title, Location, Date 41
http://sabiosciences.com/mirna_pcr_array.php
Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation
miScript miRNA PCR Arrays & Assays
Title, Location, Date 42
SampleIsolation & Preparation
Assay Set-up & Analysis
Customer support group:1-888-503-3187
Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation
Gene Expression� RT-PCR - http://sabiosciences.com/PCRArrayPlate.php� Somatic mutations - http://sabiosciences.com/somaticmutation.phpBCR and TLR signaling pathways are recurrently targeted by genetic
changes in splenic marginal zone lymphomas(MYD88 mutations) (Haematologica. 2012 April; 97(4): 595–598)� Copy Number Variations & Alterations -
http://sabiosciences.com/copynumber.phpActivation of toll-like receptors 2, 3, and 4 on human melanoma cells
induces inflammatory factors (Mol Cancer Ther 2008;7(11):3642–53)Epigenetics
� miRNA - http://sabiosciences.com/mirna_pcr_array.php� DNA methylation - http://sabiosciences.com/dna_methylation.php� Histone modifications - http://sabiosciences.com/chipqpcr.php
Transcriptional control of the inflammatory response (Nat Rev Imm, October 2009, Volume 9)Functional Studies
� Reporter assays - http://sabiosciences.com/cellassay.php� siRNA/shRNA - http://sabiosciences.com/RNAi.php
Protein Expression� Cytokine levels - http://sabiosciences.com/ELISA.php
Title, Location, Date 43
http://sabiosciences.com/home.phphttp://sabiosciences.com/seminarlist.php
Sample & Assay Technologies Keep up to date: Follow Pathway focused biology on Facebook
Latest information on pathway and disease research, resources and demos.
Sample & Assay Technologies Thank you for attending
Would you like to try a TLR & Inflammation-related PCR Array?
Title, Location, Date 45
PCR Array Starter Pack Promotion
PCR Arrays of any Pathway (FREE)• 2 96-well/100-well (2 samples)
oRT2 Profiler (mRNA): FDK-PAFAS22omiScript (miRNA): FDK-MIFAS22
Required Reagents (w/ Purchase)• RT2 First-Strand cDNA Synthesis Kit, OR• RT2 SYBR Green Mastermix (2-Pack)
Webinar-related question?
Call 1-888-503-3187 to order
Email: [email protected] and Canada only