Toll-like receptors (TLRs) play a key role in the innate immune response to pathogens and tissue damage by recognizing molecular patterns and initiating signaling pathways that induce inflammation. TLRs are expressed by immune cells like dendritic cells and activate these cells which then activate T cells and drive both innate and adaptive immune responses. MicroRNAs also regulate TLR signaling pathways and the inflammatory response by targeting mediators downstream of TLR activation. QIAGEN offers a variety of tools for studying TLR signaling and inflammation, including PCR arrays to profile gene and miRNA expression and assays for functional studies, protein analysis, and epigenetics.

1. Sample & Assay Technologies
Toll-like Receptors in Inflammation
Raed Samara, Ph.D.
R&D Scientist

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QIAGEN webinars
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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.
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Microbes/Infection
Tissue Damage
TLR
Acute Inflammation
Infection Clearance
Tissue Homeostasis
Epigenetic
Changes
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
TLR
Pre-cancer & Cancer
Chronic Inflammatory Diseases
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7. Sample & Assay Technologies
Toll-like Receptors (TLR)
Recognize molecular
patterns derived from:
- Microbes
- Endogenous molecules
Either:
- Directly
- Indirectly (with the help of
accessory molecules)
TLR8
TLR10
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
Rakoff-Nahoum & Medzhitov. 2009. Nat Rev Cancer

8. Sample & Assay Technologies
Ligands of TLR
Chen et al. 2007. Int’l Immunopharm
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9. Sample & Assay Technologies
Expression of TLR
TLR are expressed in a wide variety of cells
Ospelt & Gay. 2010. Int’l J Biochem Cell Biol
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10. Sample & Assay Technologies
TLR signaling
TAK1
Foster & Medzhitov. 2009. Clin Imm
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11. Sample & Assay Technologies
TLR signaling
Uematsu & Akira. 2007. JBC
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12. Sample & Assay Technologies
TLR signaling
Induction of 3 classes of transcription factors
Post-translational activation
of transcription factors after
TLR stimulation
Transcription factors are
induced during the primary
response
Transcription factors are cellspecific
Medzhitov & Horng. 2009. Nat Rev Imm
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13. Sample & Assay Technologies
TLR in inflammation
TLR activation leads to a complex module-specific inflammatory
transcriptional response
Medzhitov & Horng. 2009. Nat Rev Imm
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14. Sample & Assay Technologies
TLR in inflammation
TLRs form a bridge between innate and adaptive immune responses
Parker, Prince, Sabroe. 2007. Clin Exp Imm
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15. Sample & Assay Technologies
TLR in inflammation
TLR
DC
T cells
Mills. 2011. Nat Rev Imm
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17. 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 RTPCR
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18. 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
Title, Location, Date
Knockout of MYD88
decreased Th1 responses
after induction of EAE
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19. Sample & Assay Technologies
Which cell type is it?
TLR
DC
T cells
Mills. 2011. Nat Rev Imm
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20. 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
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21. Sample & Assay Technologies
TLR in inflammation
TLR
DC
T cells
Mills. 2011. Nat Rev Imm
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23. Sample & Assay Technologies
Effects of TLR activation on Treg cell activity
TLR activation in Tregs suppresses their suppressive functions
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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
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25. Sample & Assay Technologies
Regulation of TLR signaling
Foster & Medzhitov. 2009. Clin Imm
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26. Sample & Assay Technologies
QIAGEN tools for studying TLR in inflammation
RT2 Profiler PCR Arrays & Assays
Profile 84 different
genes on one array
Appropriate controls
for data normalization,
sample quality, and
reaction performance
Focus on your
questions and papers
http://sabiosciences.com/PCRArrayPlate.php
Title, Location, Date
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27. Sample & Assay Technologies
QIAGEN tools for studying TLR in inflammation
RT2 Profiler PCR Arrays & Assays
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
Title, Location, Date
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28. Sample & Assay Technologies
QIAGEN tools for studying TLR in inflammation
RT2 Profiler PCR Arrays & Assays
Sample
Isolation &
Preparation
Assay
Set-up &
Analysis
Customer support group:
1-888-503-3187
support@SABiosciences.com
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29. Sample & Assay Technologies
Regulation of TLR signaling
Quinn & O’Neil. 2011. Int’l Imm
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30. Sample & Assay Technologies
miRNA in TLR & inflammation
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miRNA webinars
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miRNA webinars
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34. Sample & Assay Technologies
Expression of miR-181 in hematopoietic lineages
Northern analyses
Chen, …, Bartel et al. 2004. Science
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Expression of miR-181 in hematopoietic lineages
Ectopic expression
Transduce bone marrow cells with Vector or miR-181; reconstitute mice with
transduced BM cells; detect cell lineages by flow
Monocytes Neutrophils nonmyeloid
Chen, …, Bartel et al. 2004. Science
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37. Sample & Assay Technologies
Expression of miRNAs after TLR activation
Microarray analyses
Induce THP-1 cells (human acute monocytic leukemia) with LPS
Microarray results are validated
by RT-PCR
Taganov, …, Baltimore et al. 2006. PNAS
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38. Sample & Assay Technologies
Expression of miR-146a is NFkB-dependent
Mutations in NFkB binding site
in the genomic loci of miR-146a
Taganov, …, Baltimore et al. 2006. PNAS
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39. 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
Taganov, …, Baltimore et al. 2006. PNAS
Title, Location, Date
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40. Sample & Assay Technologies
QIAGEN tools for studying TLR in inflammation
miScript miRNA PCR Arrays & Assays
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.php
Title, Location, Date
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41. Sample & Assay Technologies
QIAGEN tools for studying TLR in inflammation
miScript miRNA PCR Arrays & Assays
http://sabiosciences.com/mirna_pcr_array.php
Title, Location, Date
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42. Sample & Assay Technologies
QIAGEN tools for studying TLR in inflammation
miScript miRNA PCR Arrays & Assays
Sample
Isolation &
Preparation
Assay
Set-up &
Analysis
Customer support group:
1-888-503-3187
support@SABiosciences.com
Title, Location, Date
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43. QIAGEN tools for studying TLR in inflammation
Sample & Assay Technologies
Gene Expression
RT-PCR - http://sabiosciences.com/PCRArrayPlate.php
Somatic mutations - http://sabiosciences.com/somaticmutation.php
BCR 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.php
Activation 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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45. Sample & Assay Technologies
Thank you for attending
Would you like to try a TLR & Inflammation-related PCR Array?
PCR Array Starter Pack Promotion
PCR Arrays of any Pathway (FREE)
• 2 96-well/100-well (2 samples)
2 Profiler (mRNA): FDK-PAFAS22
oRT
omiScript (miRNA): FDK-MIFAS22
Required Reagents (w/ Purchase)
• RT2 First-Strand cDNA Synthesis Kit, OR
• RT2 SYBR Green Mastermix (2-Pack)
Call 1-888-503-3187 to order
Email: support@SABiosciences.com
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