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Toll-like Receptors in Inflammation

Samuel J. Rulli, Ph.D.

Samuel.Rulli@qiagen.com

Questions? Email us at:

BRCsupport@qiagen.com

QIAwebinars@qiagen.com

Part Two of a Four-Part Webinar Series on Microbiology

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A four-part webinar series on host responses

Toll-like receptors in inflammation 2

Exploring the first line of defense: research tools for the innate immune system

Toll-like receptors in inflammation

Studying the adaptive immune response: tools for T and B cell research

The crosstalk between cancer inflammation and immunity: exploring cancer immune responses

Explore host responses and defense mechanisms:

2

1

3

4

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Toll-like receptors in inflammation 3

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Agenda

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• Inflammation: the role of Toll-like receptors (TLRs)

• Toll-like receptors: an overview

• Toll-like receptors: signaling

• TLR8 in HIV expression from latent reservoirs◦ RT2 Profiler PCR Arrays

•  lncRNAs mediate immune regulation

• TLR-mediated induction of microRNAs◦ microRNAs associated with TLR signaling◦ miScript miRNA PCR Arrays

• Summary

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Inflammation: an introduction

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>Definition: a protective response to tissue damage or microbes that serves to destroy, dilute or wall off the injuring agent and the injured tissues

• Cytokines & chemokines• Signaling pathways• Immune system composition

• Epigenetic changes

• mRNA changes

Resolu

tion

Microbes/infection/

tissue damage

Acute inflammation

Infection clearance/

tissue homeostasis

Persistence

Chronicinflammation

Pre-cancer & cancer/chronic

inflammatory diseases

Toll-likereceptors

Toll-likereceptors

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Toll-like receptors: signaling

• Toll-like receptors (TLRs):◦ A family of membrane-spanning innate immune

receptors ◦ Recognize ligands derived from bacteria, fungi,

viruses, parasites and host proteins/factors via pathogen associated molecular patterns (PAMPs)

• All TLRs share a common modular structure: ◦ A leucine rich repeat (LRR)-containing ectodomain

responsible for ligand-binding◦ A membrane-spanning region◦ A cytosolic signaling domain called the

Toll-interleukin-1 receptor homology domain (TIR)

• TLRs induce the expression of genes required for: ◦ The inflammatory response, including

▬ Inflammatory cytokines▬ Chemokines▬ Antimicrobial molecules▬ Major histocompatibility (MHC)▬ Co-stimulatory molecules important for adaptive

immune activation

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Toll-like receptors: signaling

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Rakoff-Nahoum, S., and Medzhitov, R. (2009) Toll-like receptors and cancer. Nat Rev Cancer 9, 57-63

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

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Ligands of TLRs – host factors can be ligands too!

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Chen, K., and Huang, J., et al. (2007) Toll-like receptors in inflammation, infection and cancer. Int. Immunopharmacology, 7, 1271

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Toll-like receptors: expression

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Ospelt, C., and Gay, S. (2010) TLRs and chronic inflammation. Int. J. Biochem. Cell Biol. 42, 495

TLRs are expressed in a wide variety of cells

TLR Expression

TLR1 Monocytes, macrophages, B cells, T cells, DCs, PMN, NK cells, non-immune cells (fibroblasts, astrocytes, epithelial cells, keratinocytes)

TLR2 Monocytes, macrophages, DCs, PMN, non-immune cells (fibroblasts, astrocytes, epithelial cells, keratinocytes)

TLR3 DCs, macrophages, mast cells, NK cells, non-immune cells (fibroblasts, astrocytes, epithelial cells, keratinocytes)

TLR4 Monocytes, macrophages, DCs, PMN, non-immune cells (fibroblasts, astrocytes, epithelial cells, keratinocytes)

TLR5 Monocytes, macrophages, T cells, DCs, PMN, non-immune cells (fibroblasts, astrocytes, epithelial cells, keratinocytes)

TLR6 Monocytes, macrophages, B cells, T cells, DCs, PMN, NK cells, non-immune cells (fibroblasts, astrocytes, epithelial cells, keratinocytes)

TLR7 B cells, plasmacytoid DCs

TLR8 Monocytes, myeloid DCs

TLR9 B cells, plasmacytoid DCs, GI epithelial cells, keratinocytes

TLR10 B cells, plasmacytoid DCs

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Toll-like receptor signaling: transcriptional control

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Post-translational activationof transcription factors after TLR stimulation

Transcription factors are induced during the primary response

Transcription factors are cell-specific

Induction of three classes of transcription factors

Medzhitov, R., and Horng, T. (2009) Transcriptional control of the inflammatory response. Nat. Rev. Immunol. 9, 692

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TLRs in inflammation

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Activation of TLRs leads to a complex, module-specific, inflammatory transcriptional response based on the pathways activated

Medzhitov, R., and Horng, T. (2009) Transcriptional control of the inflammatory response. Nat. Rev. Immunol. 9, 692

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Parker, L.C., Prince, L.R., and Sabroe, I. (2007) Translational mini-review series on Toll-like receptors: networks regulated by Toll-like receptors mediate innate and adaptive immunity. Clin. Exp. Immunol. 147, 199

TLRs in inflammation: linking innate and adaptive immune responses

TLRs form a bridge between innate and adaptive immune responses

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TLRs are associated with diverse diseases

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Inflammation Allergy Brain Ischemia

Cancer Diabetes Mellitus Myocarditis

Infection Metastasis Pneumonia

Injury Insulin Resistance Septic Shock

Mutation Tuberculosis Brain Injury

Atherothrombosis Rheumatoid Arthritis Pain

Breast Tumor Prostate Cancer Lung Cancer

Anoxia Stroke Systemic Lupus Erythematosus

Septicemia Cirrhosis Necrosis

Death Myocardial Infarction Thrombosis

Ischemia Periodontitis

Autoimmunity Large Cell Lymphoma

Cardiac Failure Squamous Cell Carcinoma

Autoimmune Disease Infectious Disease

Arthritis Bacterial Infection

Reperfusion Injury Lupus

Hepatobiliary Disease/Toxicity Melanoma

Asthma Acne Vulgaris

Colitis Type 2 Diabetes Mellitus

Type 1 Diabetes Mellitus Stomach Cancer

https://www.qiagen.com/shop/genes-and-pathways/knowledgehub/pubmed-publications/

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Research solutions at QIAGEN

Toll-like receptors in inflammation

miRNA PCR ArraysGene expression lncRNA PCR Arrays

Pathway-focused approach

lncRNA PCR Arrays

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TLR signaling RT2 Profiler PCR Array: an application example

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TLR8 HIV

?

How does TLR8 signaling lead to latent HIV production from myeloid-monocytic cells and from CD4 T-cells?

www.jimmunol.org/content/186/7/4314.long

Myeloid-monocytic

cells

CD4 T cells

HIV

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QIAGEN tools for studying TLRs in inflammation

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RT2 Profiler PCR Arrays & Assays

Sample Isolation & Preparation

Assay Set-up & Analysis

Culture cells with cells isolated from blood samples of 6 HIV+ patients

RT2 TLR Signaling PCR Array

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QIAGEN tools for studying TLRs in inflammation

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• Profile 84 different genes using one array

• Appropriate controls for◦ data normalization◦ sample quality ◦ reaction performance

• Allows you to focus on your questions and papers

Gene expression profiling - RT2 Profiler PCR Arrays & Assays

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Toll-like Receptor Signaling PCR Array

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Toll-Like Receptor Signaling Pathway RT2 Profiler PCR Array

https://www.qiagen.com/usearch/rt2-profiler-pcr-arrays?catno=PAHS-018Z#geneglobe

Guaranteed system

• Relevance

• Sensitivity

• Reproducibility

• Specificity

• Uniform PCR

amplification efficiency

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TLR8

Toll-like receptors: signaling

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Foster, S.L., and Medzhitov, R. (2009) Gene-specific control of the TLR-induced inflammatory response. Clin. Immunol. 130(1), 7

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Toll-like receptors: signaling

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Using RT2 Profiler TLR PCR Arrays to analyze pathway activity

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TLR8

Toll-like receptors: signaling

Toll-like receptors in inflammation 21

Foster, S.L., and Medzhitov, R. (2009) Gene-specific control of the TLR-induced inflammatory response. Clin. Immunol. 130(1), 7

Further activation of latent

HIV in CD4 T-cells

TNF-alpha

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QIAGEN tools for studying TLRs in inflammation

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TLR signaling pathway Antibacterial responses

NFkB signaling pathway Antiviral responses

NFkB signaling targets Antifungal responses

MAPK signaling pathway Inflammatory responses and autoimmunity

PI3K-Akt signaling pathway Innate and adaptive immune responses

Th1 & Th2 responses Cytokines & chemokines

Interferons & receptors Over 170 other pathways

Inflammasomes 13 species

Available for: human, mouse, rat, monkey, dog, zebrafish, rabbit, pig, fruit fly, chicken, horse, cow and CHO cells

RT2 Profiler PCR Arrays & Assays

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Research solutions at QIAGEN

Toll-like receptors in inflammation

miRNA PCR ArrayslncRNA PCR ArraysGene expression

Pathway-focused approach

lncRNA PCR Arrays

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lncRNAs: new players in human diseases

Toll-like receptors in inflammation 24

• lncRNAs are involved in gene regulatory networks that govern host-pathogen interactions

• Toll-like receptors induce the expression of lncRNAs

• lncRNA-Cox2 mediates both the activation and repression of distinct classes of immune genes

• lncRNAs are involved in the control of NFkB signaling and inflammation

• PACER controls Cox-2 expression by binding the repressive NF-kB subunit p50

Carpenter, S., et al. (2013) A Long Noncoding RNA Mediates Both Activation and Repression of Immune Response Genes. Science. 341, 789Krawczyk, M., and Emerson, B.M. (2014) p50-Associated COX2 Extragenic RNA (PACER) activates COX-2 gene expression by occluding repressive NF-κB complexes. Elife. 3:e01776

• lncRNAs play important roles in a wide range of biological processes • Dysregulated lncRNAs are involved in many complex human diseases.

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Molecular functions of lncRNAs

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Martin, L., Chang, H.Y. (2012) Uncovering the role of genomic "dark matter" in human disease. J Clin. Invest. 122, 1589

lncRNAs are a novel class of RNAs, larger than 200 nucleotides

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QIAGEN tools for studying TLRs in inflammation

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• lncRNA databases

◦ In-house databases at QIAGEN GeneGlobe

◦ Provide more than 40,000 human and 27,000 mouse lncRNA assays

• RT2 lncRNA Assays◦ Laboratory-verified for optimal qPCR performance – high specificity,

amplification efficiency and sensitivity

• RT2 lncRNA qPCR Arrays

◦ Pathway or disease-relevant lncRNA assays

◦ RT2 lncRNA Inflammatory Response and Autoimmunity Array and RT2 lncFinder PCR Array (Human & Mouse)

• Custom options◦ Flexible custom designs from the lncRNA and qPCR databases to

profile mRNAs and lncRNAs simultaneously

• lncRNA isolation

◦ miRNeasy and exoRNeasy kits

• Free on-line data analysis tool

http://www.qiagen.com/us/landing-pages/lncrna/

RT2 lncRNA PCR Arrays and Assays

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Experiment design: discover lncRNAs in your total RNA samples

Toll-like receptors in inflammation 27

2. cDNA synthesis: convert RNA to cDNA.

3. Pre-amplification with RT2 PreAMP cDNA kit.

1. Total RNA isolation.

4.

5.

Samples: control and treated • Total RNA isolation: isolate total RNA with miRNeasy Kits• cDNA synthesis: convert total RNA to cDNA using RT2 First

Strand Kit• Pre-amplification: Pre-amplify the target lncRNAs with

pre-amplification RT2 PreAMP Primer mixes (optional)• Run PCR: detect lncRNAs with RT-PCR

◦Human RT2 lncRNA Cancer PathwayFinder PCR Array

◦Master mix: RT2 SYBR® Green qPCR Mastermix

◦qPCR cyclers

• Free data analysis: GeneGlobe Data Analysis Center

RT2 lncRNA PCR Array Inflammatory Response & Autoimmunity

84 lncRNAs involved in:• Acute-Phase Response• Autoimmunity• Humoral Immune Response• Inflammatory Response• Innate & Adaptive Immunity• Other Inflammatory Response & Autoimmunity

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Regulation of TLR signaling through miRNAs

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Quinn, S.R., and O’Neil, L.A. (2011) A trio of microRNAs that control Toll-like receptor signalling. Int. Immunol. 23, 421

miRNAs can be • upregulated by TLR signaling AND• feedback and regulate TLR pathway responses

miR-155 TargetsmiR-146a TargetsmiR-21 Targetslet-7 Targets miR-1 and miR-206 TargetsmiR-153 Targets

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Research solutions at QIAGEN

Toll-like receptors in inflammation

lncRNA PCR Arrays miRNA PCR ArraysGene expression

Pathway-focused approach

lncRNA PCR Arrays

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miRNA biogenesis – like a gene or part of exon/intron

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• Transcribed by RNA polymerase II as long primary transcripts (pri-miRNAs), which may contain more than one miRNA

• In the nucleus, pri-miRNAs are processed to hairpin-like pre-miRNAs by RNAse III-like enzyme Drosha

• Pre-miRNAs are then exported to the cytosol by Exportin 5

• In the cytosol, RNAse III-like Dicer processes pre-miRNAs into mature miRNAs

• Mature miRNAs are incorporated into RISC

• miRNAs with high homology to target mRNAs lead to mRNA cleavage

• miRNAs with imperfect base pairing to target mRNAs lead to translational repression and/or mRNA degradation

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miRNA array application: miR-146 in negative feedback loops

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Application example:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1567904/

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Expression of miRNAs after activation of TLRs

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Microarray results are validated by RT-PCR

Induce THP-1 cells (human acute monocytic leukemia) with LPS

Microarray analyses

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Expression of miR-146a is NF-κB-dependent

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

Taganov, K.D., Boldin, M.P., Chang, K.J., and Baltimore, D. (2006) NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. PNAS 103, 12481

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miR-146 targets mediators downstream of TLR

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Transfect cells with TRAF6 or IRAK1 reporter constructs

Transfect cells with miRNAs

Read luciferase activity

http://www.ncbi.nlm.nih.gov/pubmed/16885212

miR-146a Targets PCR Arrays

Taganov, K.D., Boldin, M.P., Chang, K.J., and Baltimore, D. (2006) NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. PNAS 103, 12481

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microRNAs associated with TLR signaling

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miR-146a miR-146 miR-132 let-7e miR-143

miR-155 miR-145 miR-29c miR-181a miR-212

let-7i miR-210 miR-107 miR-302b miR-1915

miR-34c miR-27b miR-31 miR-16 miR-34a

miR-147 miR-574 miR-1225 miR-187 miR-466l

miR-125a miR-92a miR-146b miR-128a miR-149

miR-21 miR-202 miR-200c miR-200a miR-7

miR-26a miR-29b miR-200b miR-221 miR-23a

miR-125b miR-301a miR-126 miR-10a miR-223

let-7b miR-9 miR-17 miR-142 miR-150

https://www.qiagen.com/shop/genes-and-pathways/knowledgehub/pubmed-publications/

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QIAGEN tools for studying TLRs in inflammation

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miScript miRNA PCR Arrays• miRNome

◦ Human: miRBase v21, covers 2,402 primer assays◦ Mouse: miRBase v21, covers 1,765 primer assays◦ Rat: 653 primer assays◦ Dog: 277 primer assays◦ Rhesus macaque: 469 primer assays◦ Cow: 744 primer assays

• Pathway-focused arrays (>20 arrays)◦ miFinder◦ Inflammatory Response◦ Autoimmunity◦ T-Cell & B-Cell Activation ◦ Immunopathology◦ Cell Differentiation & Development◦ Serum & Plasma◦ Apoptosis

miScript PreAMP Kit• Optional step for small or precious samples• Full miRNome profiling from as little as 1 ng RNA

•Pre-formatted, single-use PCR arrays with wet-lab verified assays• miRNA function can be analyzed using microRNA mimics, inhibitors and target

protectors

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QIAGEN tools for studying TLRs in inflammation

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miScript miRNA PCR Array workflow1. Isolate total RNA with miRNeasy kits

2. Perform reverse-transcription

3. Prepare PCR pre-mix

4. Load arrays & perform real-time PCR

5. Analyze Data

1 hour

2 minutes

2 hours

5 minutes

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QIAGEN tools for studying TLRs in inflammation

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• Profile 84 different miRNAs using one array

• Appropriate controls for◦ data normalization◦ sample quality ◦ reaction performance

• Allows you to focus on your questions and papers

miScript miRNA PCR Arrays

http://www.qiagen.com/search/miscript-mirna-pcr-arrays

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QIAGEN TLR signaling research portal

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https://www.qiagen.com/shop/genes-and-pathways/complete-biology-list/toll-like-receptor-signaling/

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QIAGEN Toll-Like Receptor Signaling Research Solutions

Solutions & Technologies• RT2 Profiler PCR Array

• RT2 lncRNA PCR Array

• miScript miRNA PCR Array

• EpiTect Methylation Arrays

• EpiTect Chromatin IP

• siRNA and shRNA

• Mutation analysis

• Multi-Analyte ELISArry Kits

• Cignal Reporter Assays

https://www.qiagen.com/shop/genes-and-pathways/complete-biology-list/toll-like-receptor-signaling/

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Questions?

41

Samuel J. Rulli, Ph.D.

Samuel.Rulli@qiagen.com