Tlr and inflammation 2013

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Toll-like Receptors in InflammationRaed Samara, Ph.D.R&D Scientist

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Sample & Assay Technologies 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

mRNAChangesTLR

Sample & Assay Technologies Toll-like Receptors (TLR)

Rakoff-Nahoum & Medzhitov. 2009. Nat Rev Cancer

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

Chen et al. 2007. Int’l Immunopharm

Sample & Assay Technologies Expression of TLR

TLR are expressed in a wide variety of cells

Ospelt & Gay. 2010. Int’l J Biochem Cell Biol

Sample & Assay Technologies TLR signaling

Foster & Medzhitov. 2009. Clin Imm

Uematsu & Akira. 2007. JBC

Induction of 3 classes of transcription factors

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

Medzhitov & Horng. 2009. Nat Rev Imm

TLRs form a bridge between innate and adaptive immune responses

Parker, Prince, Sabroe. 2007. Clin Exp Imm

Mills. 2011. Nat Rev Imm

DC T cells

Sample & Assay Technologies Expression of TLR

Expression of TLR and MYD88 in the CNS of EAE-diseased mice

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

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?

DC T cells

Sample & Assay Technologies Effects of modulating TLR activity on EAE

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

DC T cells

Sample & Assay Technologies Effects of TLR activation on Treg cell activity

TLR activation in Tregs suppresses their suppressive functions

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

Knockout indicated components in Tregs, sort Tregs, co-culture sorted Tregs with naïve CD4+ cells

Sample & Assay Technologies Regulation of TLR signaling

Foster & Medzhitov. 2009. Clin Imm

Sample & Assay Technologies QIAGEN tools for studying TLR in inflammation

RT2 Profiler PCR Arrays & Assays

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

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

SampleIsolation & Preparation

Assay Set-up & Analysis

Customer support group:1-888-503-3187

support@SABiosciences.com

Sample & Assay Technologies Regulation of TLR signaling

Quinn & O’Neil. 2011. Int’l Imm

Sample & Assay Technologies miRNA in TLR & inflammation

Sample & Assay Technologies miRNA webinars

Sample & Assay Technologies Expression of miR-181 in hematopoietic lineages

Northern analyses

Chen, …, Bartel et al. 2004. Science

Sample & Assay Technologies Expression of miR-181 in hematopoietic lineages

Ectopic expression

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 Expression of miRNAs after TLR activation

Microarray analyses

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

Mutations in NFkB binding site in the genomic loci of miR-146a

Sample & Assay Technologies miR-146 targets mediators downstream of TLR

Transfect cells with TRAF6 or IRAK1 reporter constructs, transfect cells with miRNAs, read luciferase activity

miScript miRNA PCR Arrays & Assays

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

http://sabiosciences.com/mirna_pcr_array.php

SampleIsolation & Preparation

Assay Set-up & Analysis

Customer support group:1-888-503-3187

support@SABiosciences.com

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

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Tlr and inflammation 2013

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