Exploring the First Line of Defense - Research Tools for Innate Immnity: Host Defense Webinar Series Part 1

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Exploring the First Line of Defense:
Research Tools for the Innate Immune System
Miranda Hanson-Baseler, PhD
Miranda.Hanson-Baseler@qiagen.com
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A four-part webinar series on host responses
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:
1
2
3
4

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Legal disclaimer
3
QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention or treatment of a disease.
For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available at
www.QIAGEN.com or can be requested from QIAGEN Technical
Services or your local distributor.

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Innate vs adaptive immunity
• PAMPs/DAMPs
• Rapid induction (hours)
• Short-lived
• Antigenic peptides
• Slow induction (days)
• Memory
Innate immunity Adaptive immunity
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Innate immune response
Recognition
• Recognize microbial
components
• Germline-encodedreceptors
• Non-clonal (i.e., all cells of
the same lineage have same
receptors)
• Macrophage recognition of
pathogens  inflammation
• Inflammation leads to PMN,
monocyte (M/DC), NK and
eosinophil recruitment via
chemokines
• Non-cytokines or
chemokines (complement
fragments, fMLP) recruit and
activate innate immune cells
• Phagocytosis and microbial
killing by PMN and M
• Cytotoxicity by NK cells
• Induction of adaptive
immunity by DC and M
Phases of the innate immuneresponse
EffectorRecruitment/Activation

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Epithelia, peptides, innate-like lymphocytes
Microbial invaders enter the body through skin and mucosa, and are met by several
innate defenses before they encounter circulating cellular effectors:
• Mucus/cilia (sweep out microbes before they can adhere)
• Chemical defenses
◦ b-defensins (epithelial cells and leukocytes, skin, tongue, respiratory tract)
◦ a-defensins (Paneth cell granules in intestine, neutrophil granules)
◦ Lysozyme, phospholipase A (saliva, tears)
◦ pH and digestive enzymes (stomach)
Innate-like lymphocytes are lymphocytes with limited receptor diversity:
• Intraepithelial g/d T lymphocytes (GI)
• B-1 B cells (peritoneal and pleural cavities)
• NK-T cells (thymus, peripheral lymphoid organs)
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Induction of innate immunity by PRRs
Pathogen-associated molecular patterns (PAMPs)
• Lipopolysaccharides (LPS)
• Flagellin
• Single- and double-stranded nucleic acids
• CpG
• Lipoteichoic acid
• Zymosan
• Others
Danger-associated molecular patterns (DAMPs)
• HMGB1
• Heat shock proteins
Pattern recognition receptors (PRRs)
• Toll-like receptors (TLRs)
• NOD-like receptors (NLRs)
• Mannose receptor
• Cytosolic DNA sensors
• RIG1-like receptors (RLRs)
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Cellular and molecular effectors of innate immunity
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Phagocytes and natural killer (NK) cells
Macrophages
• Long-lived phagocytes
• Mannose receptor,
scavenger receptors
• Complement receptors
• ROS, RNI
• Produce proinflammatory
cytokines (IL-1b, IL-12, TNF-
a, IL-6,CXCL8)
• Produce growth factors for
tissue remodeling
• Arise from circulating
monocytes
• Short-lived phagocytes
• Granules (acid hydrolase,
myeloperoxidase, defensins,
cathepsin G, lysozyme,
lactoferrin, elastase, etc)
• Respiratory burst
• NETs (neutrophil
extracellular traps:
chromatin + serine
proteases)
• Proinflammatory cytokines
(IL-12, TNF-a)
• Cytotoxic cells (via perforin
and granzymes, or ADCC)
• Expansion/activation in
response to IL-15, IL-12,
Type I IFNs
• Produce IFN-g, IL-1, and IL-2
• Recognize Class I MHC
(inhibitory & activating
receptors)
• Blood, spleen localization
• Memory?
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Neutrophils NK cells

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EosinophilsBasophilsMast cells Nuocytes
• Helminths, viruses
and bacteria (also
allergies)
• Respond to
complement, IgE,
TLR stimulation
• Release
histamine,
heparin,
proteases, TNF
and other
proinflammatory
cytokines,
eicosanoids, etc.
• Least abundant
granulocyte
• Histamine,
proteoglycans,
lipid mediators,
IL-4, IL-17E
• Macroparasite
and helminth
defense, allergy
• IgE-activated,
matures via IL-3
• Important in Type
2 innate response
against helminth
infection(1)
• Respond to IL-25
and IL-33,
produce IL-13
• Involved in
allergic lung
inflammation
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• Produce major basic
protein, eicosanoids,
histamine,
peroxidases, acid
phosphatase,
growth factors, and
cytokines
• Helminths, viral
infections, allergies
• Activated by IL-5, IL-
3, and GM-CSF
(1) Neill, D.R. et al. (2010) Nuocytes represent a new innate effector leukocytethat mediates type-2 immunity. Nature 464, 1367-1370

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Dendritic cells
• “Professional antigen presenting cells (APCs)” – link innate and adaptive immunity
(macrophages and B cells are also considered professional APCs)
• Present as immature DCs (highly phagocytic) in tissues; upon antigen capture, migrate
to lymph nodes and spleen and mature, providing Ag presentation and co-stimulation to
T cells
• Multiple subsets and sub-subsets based on surface markers, localization and function
• Produce IL-12, IL-15, TNF-a and Type I IFNs, and respond to chemokines through
CCR2, CCR5, CCR6 and CCR7
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Complement cascade
Three types:
• Classical
• Alternative
• Lectin-induced
Initiation:
• C1q, mannose-bindinglectin or C3b bind
microbe surfaces (or C1q binds C-reactive
protein)
• All three pathways lead to cleavage of C3
Outcomes:
• Opsonization
• Chemotaxis/activation of leukocytes (C3a, C5a)
• Membrane-attack complex
◦ Results from C3b generation
◦ Consists of C5b-9
◦ Effective against Neisseriaspecies
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Cytokines and chemokines
Cytokines produced by innate immune cells:
• Type I IFNs, IFN-g
• TNF-a
• IL-1, 6, 8, 10, 12, 15, 18
Cytokines that enhance/inhibit innate immune
activity:
• Enhance: IFN-g, TNF-a, IL-15, IL-12
• Inhibit: IL-10, TGF-beta
Important chemokine receptors:
• CXCR1, 2 (neutrophils)
• CCR1, 2, 3 (leukocytes)
• CCR5 (DCs, NK cells, monocytes)
• CCR6, 7 (DCs)
• CXCR3 (ligand: CXCL10, monocytes and NK cells)
Important chemokines:
• CXCL8 (produced by M, recruits PMN)
• CCL2 (produced by monocytes and fibroblasts, and
recruits monocytes, DCs, NK cells)
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Important signaling networks
Cytokines
• JAK/STAT
• MAPK
• IRFs
• PI3K
• NFκB
Chemokines
• PI3K/AKT
• MAPK
• NFκB
TLRs
• NFκB
• JNK/MAPK
• IRFs
• PI3K
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Outcomes of an innate immune response
Microbial killing
• Complement
• Neutrophil and macrophage microbicidal mechanisms
• NK cell cytotoxicity against infected cells
• Type I IFNs induce antiviral state in infected cells
Induction of adaptive immunity
• Dendritic cells and macrophages present antigens to T cells and provide co-stimulation
• Chemokine and cytokine production leads to lymphocyte expansion and activation
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Technologies for innate immune research
Pathway-focused gene expression analysis
• RT2 Profiler PCR Arrays
Signaling pathway reporter arrays
• Cignal Finder Reporter Arrays
Cytokine analysis
• Multi-Analyte ELISArrays
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Pathway-focused gene expressionanalysis
• Featured publication: Derbigny, W.A. et al., 2012(1)
• RT2 Profiler PCR Array: Mouse Inflammatory Cytokines
• Featured publication: Zughaier, S.M., 2011(2)
• Cignal Finder 10=Pathway Reporter Array
• RT2 Profiler PCR Array: Human TLR and Human
Apoptosis
Cytokine analysis
• Featured publication: Rahman, S. et al., 2011(3)
• Multi-Analyte ELISArray
• RT2 Profiler PCR Array: Mouse IFN-a and IFN-b
Response, Mouse Dendritic and Antigen Presenting Cells
(1) Derbigny, W.A. et al. (2012) Identifying a Role for Toll-Like Receptor 3 in the Innate Immune Response to Chlamydia muridarum Infection in Murine
Oviduct Epithelial Cells. Infect Immun. 80, 254-265
(2) Zughaier, S.M. (2011) Neisseria meningitidis capsular polysaccharides induce inflammatory responses via TLR2 and TLR4-MD-2. Leuk Biol. 89, 469-480
(3) Rahman, S. et al. (2011) Role of Th1, Th2 and Th17 cytokines on the expression of various inflammatory cytokines, defensive proteins and chemokines
by primary human epithelial keratinocytes. J Immunol. 186, 148

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RT2 Profiler PCR Arrays
• Assay 84 of the most relevant genes in biological and disease pathways
• Gene lists identified through state-of-the-art bioinformatics and text-mining tools
• Integrated controls for genomic DNA contamination, normalization and PCR processes
• Web-based data analysis software at no additional cost
• Compatible with most real-time PCR instruments
Immunity-related pathways:
• Innate & adaptive immune response
• Inflammatory cytokines & receptors
• Dendritic & antigen-presenting cells
• Inflammasomes
• IFN-a/b response
• NFkB signaling
• MAPK signaling
• PI3K/AKT signaling
(and more… 170+ pathways, including
custom arrays)
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Application data
Experimental question: which cytokines alter expression after PMA-ionomycin treatment?
Human PBMCs
• Were treated with PMA and ionomycin
• Analyzed using the Common Cytokines
RT2 Profiler PCR Array
This volcano plot
• Shows both fold-change and the
statistical significance
• Demonstrates that, in response to
treatment
◦ 23 genes, including IL-10, IFN-
gamma, IL-2 and TNF were
upregulated,
◦ 6 genes, including IL-1beta, were
downregulated
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How does gene expression change during infection?
Case study #1
Characterizing overall responses of gene networks – as opposed to just a few genes –
can provide a more comprehensive picture of the changes that are occurring.
How can you profile the most relevant genes to your system of interest all at once?
Context:
• Research aim:to determine how oviduct epithelial cells participate in host defense against
Chlamydia
• Technique: used Mouse Inflammatory Cytokines RT2 Profiler PCR Array to compare gene
expression in infected wild-type or TLR3-deficient OE cells
• Significance:demonstrated a role for TLR3 and IFN-b in initiating inflammatory responses
against an intracellular bacterial pathogen.
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Case study #1: experimental design and results
Compared gene expression changes using the Mouse Inflammatory Cytokines PCR
Array under the following conditions :
• Wild-type cells plus C. muridarum
• TLR3-deficientcells plus C. muridarum alone
• TLR3-deficientcells plus IFN-b and C. muridarum
• TLR3-deficientcells plus IFN-b alone
Results:
• Wild-type cells plus C. muridarum: increase in most chemokines and interleukins,
decrease in CXCL15, CCR10.
• TLR3-deficient cells plus C. muridarum alone: diminished response to infection, except
CXCL15 effects; CCR9 and Lta showed modest increase compared to WT cells.
• TLR3-deficient cells plus IFN-b and C. muridarum: partially rescued chemokine and
Casp1 responses
• TLR3-deficient cells plus IFN-b alone: still stimulated some chemokine response, but not
as much as in cells also infected with C. muridarum
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Case study #1: conclusions
• The team concluded that TLR3 and IFN-b are major mediators of the inflammatory
response to C. muridarum in OE cells, possibly in a cell-type-specific manner (as other
studies had showed no involvement of TLR3 in the macrophage response).
• The ability to easily compare expression of many pathway-related genes in several different
conditions permitted the team to dissect the specific roles of the receptor, the cytokine and
the pathogen in stimulating responses.
• The CXCL10 findings from the PCR Array, as well as ELISA for CXCL10 and IL-6, prompted
the team to examine whether IFN-beta was exerting its effects through enhancement of
TLR2 signaling. Indeed, IFN-beta caused upregulation of TLR2 expression in OE cells.
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RT2 Profiler PCRArrays provide an excellent starting place for identifying
which genes in a specific biological pathway are affectedby infection,
cytokine stimulation and gene knockdown.

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• Cignal Finder 10-Pathway Reporter Array
Apoptosis
Cytokine analysis

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Cignal Reporter Assays & Arrays
Functionally verified assays for 45
pathways:
• Type I IFN
• IFN-g
• NFkB
• MAPK
• PI3K/AKT
• STAT3
• TGF-b
• And more…
Cignal Finder 10-Pathway Arrays:
• Immune Signaling
• Cancer
• Development
• Stem Cell & Differentiation
• Nuclear Receptors
• Stress & Toxicity
Cignal 45-Pathway Array
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Cignal 45-PathwayArray
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Application data
Determining the signaling pathways activated in response to cytokine stimulation
• HeLa cells were reverse
transfected with the
Immune Response 10-
Pathway Cignal Finder
Reporter Array
• 16 hours after
transfection, medium was
changed to assay
medium
• 32 hours after
transfection, cells were
treated with 5 ng/ml
TNFα or left untreated
• After 6 hours treatment,
dual-luciferase assays
were performed
• Results are expressed as
fold change
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Which signaling pathways are being triggered?
Several signaling pathways can produce innate immune responses.
How do you know which are at work in your system?
Context:
• Research aim:determining how CPS-triggered signalingproceeds
• Human TLR Signaling and Human Apoptosis RT2 Profiler PCR Arrays
• Cignal Finder 10-Pathway ReporterArray to identify active signaling pathways
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Case study #2

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Case study #2: experimental context
Pathways interrogated:
• NFkB
• PKC/Ca++ (NFAT)
• Type I IFNs (ISRE)
• IFN-g (GAS)
• MAPK/ERK (SRE)
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Context:
• Meningococcal endotoxin (LOS) produces a strong innate immune response through TLR4,but
the innate response to another crucial virulence factor– capsular polysaccharide(CPS)– was
uncharacterized
• TLR2 and TLR4-MD2were identified as the CPS receptors on macrophages
• TLRs 2 and 4 can signal through NFkB or MAPK pathways. Which pathway for CPS response?
• Purified CPS from LOS-deficientstrain of N. meningitidis (IpxA mutant)
• Dosed HEK/TLR2/6or HEK/TLR4-MD2-CD14cells with CPS or LOS after transient reverse
transfection with reporters
• Stimulated THP-1 cells with CPS-lpxA, LOS, or Rhizobium LPS (TLR4 ligands) to compare
gene induction profiles
• MAPK/JNK (AP-1)
• TGF-b (SMAD)
• cAMP-PKA (CRE)
• C-EBP
• Glucocorticoid receptor(GRE)

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Case study #2: findings and conclusions
Findings:
• Strong NFkB reporter activity in TLR4-MD2 and TLR2-6 stably transfectedcells stimulated with
serogroup B CPS
• Milder activity from Type I IFN/ISRE (TLR2-6), MAPK-JNK/AP-1 (both), and MAP-ERK/SRE
(TLR4-MD2)
• Also observed variation in gene expression profiles between cells stimulated with CPS-lpxAvs
LOS (including TNF, NOD1, CD40LG, LTA, and CARD6)
Conclusions:
Cignal Finder 10-Pathway ReporterArray identified 4 inflammatory
signal transduction pathways potentially involved in TLR-mediated
recognition of CPS
The RT2 Profiler PCRArray determined that CPS induces qualitatively
distinct gene expressionresponses compared to LOS
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• Cignal Finder 10=Pathway Reporter Array
Apoptosis
Cytokine analysis

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Multi-Analyte ELISArrays
Multi-Analyte ELISArray Kits:
• Common Cytokines
• Inflammatory Cytokines
• TLR-induced CytokinesI: Viral
• TLR-induced CytokinesII: Microbial
• Autoimmunity
• Th1 / Th2 / Th17 Cytokines
• Common Chemokines
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Application data
Experimental question: are Th1 or Th2 cytokines being produced?
• Time-dependent (0, 6, 18, 24, and 48 h) patterns of Th1/Th2 cytokine
induction by human PBMCs in response to PMA (50 µg/ml) and
ionomycin (1 µg/ml) were monitored
• The relative amount of each cytokine was profiled simultaneously using
the ELISArray Kit
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What comprises the cytokine milieu?
Innate immune responses produce a multitude of cytokines playing distinct roles.
Is it possible to efficiently detect several related cytokines simultaneously?
Context:
• Research aim:to clarify how DCs respond to HTLV-1 infection
• RT2 Profiler PCR Arrays for IFNa/b Response, Dendritic & Antigen-Presenting Cells
• Multi-Analyte ELISArray Kit to identify production of multiple cytokines at once in the context
of infection
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Case study #3

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Case study #3: background and experimental design
Study background:
• Innate immunity was not thought to have a major part in HTLV-1 control.
• However, the authors demonstrate that Flt3L-generatedDC and Type I IFN have a significant role in
controlling cell-free virus (the type that would be encountered early during infections)
Which cytokines are produced in early HTLV-1 infection?
• Cultured Flt3L-generated BMDC with cell-free virus to analyze cytokines
• Compared cytokine induction between UV-irradiated and competent virus
• Profiled gene expression upon DC infection with competent virus
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Cytokines detected:
• IL-2
• IL-4
• IL-5
• IL-6
• IL-10
• IL-12
• IL-13
• IL-17A
• IL-23
• IFN-g
• TNF-a
• TGF-b1

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Case study #3: findings and conclusions
Findings
• Multi-Analyte ELISArray: proinflammatory cytokines were secreted by DCs after viral challenge (IL-
6, TNF-a, IL-12).
• Th2 cytokines (IL-4, IL-5, IL-13) were not induced, but IL-10 and TGF-b were secreted as well
RT2 Profiler PCR Arrays
• IFNa, IFNb Response: several IFN-responsive genes were upregulated after viral challenge, as well
as signaling molecules
• DC & Antigen Presenting Cells: Some cytokines and chemokines were upregulated, confirming the
ELISArray results, and signaling and antigen uptake/presentationgenes were also enhanced. Many
key chemokines (CCL2, 7, 8) and receptors (CCR5, CCR3) were downregulated
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Conclusions:
Multi-Analyte ELISArray Kits allowed detection of multiple cytokines at
once, profiling the cytokine response of innate immune cells (in this case,
DC) to virus
RT2 Profiler PCRArrays confirmed the ELISA results and shed light on
which chemokines and IFN-responsive genes were being activated or
deactivated in response to virus

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Summary
• The innate immune system comprises of a complex network of cellular and molecular
components
• Research tools that allow simultaneous analysis of many immunity-related players at
once are an effective way to characterize innate responses to microbes
• RT2 Profiler PCR Arrays profile expression of 84 or 370 genes simultaneously and are
available for over 170 pathways. Many of these are related to innate immunity and host
defense; custom arrays are also available
• Cignal Finder Reporter Arrays (10-Pathway and 45-Pathway) permit simultaneous
cell-based reporter analysis of several signaling pathways through DNA-based or
lentiviral vectors using either GFP or luciferase
• ELISArrays are multiplex cytokine or chemokine analysis assays using a traditional
ELISA format. Custom Mix-n-Match Multi-Analyte ELISArray Kits also available
How can I learn more about researchtools for the innate immune response?
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GeneGlobe – Your new research companion!
www.qiagen.com/geneglobe
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Browse by Research Areas – Immunology
• eBiology Stores
• Cytokines & Chemokines
• Dendritic & Antigen
Presenting Cell
• Inflammatory Response &
Autoimmunity
• Innate & Adaptive Immune
Responses
• NFkB Signaling
• Toll-like Receptor Signaling
• And more!
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Contact us!
If you are interested in trying out these technologies in your research, please visit:
http://www.qiagen.com/search/rt2-profiler-pcr-arrays
http://www.qiagen.com/search/cignal-finder-reporter-arrays
http://www.qiagen.com/search/multi-analyte-elisarray-kits
When you’re ready, contact us at BRCsupport@qiagen.com to ask about our special
RT2 Profiler PCR Array starter pack offer!
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Thank you for attending today’s webinar!
Questions?
40
Miranda Hanson-Baseler, PhD
Miranda.Hanson-Baseler@qiagen.com

Sample to Insight
A four-part webinar series on host responses
41
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:
1
2
3
4

Exploring the First Line of Defense - Research Tools for Innate Immnity: Host Defense Webinar Series Part 1

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Exploring the First Line of Defense - Research Tools for Innate Immnity: Host Defense Webinar Series Part 1