1. Exploring the first line of defense:
research tools for the innate immune system
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2. Innate and adaptive immunity
Innate
Adaptive
PAMPs/DAMPs
Rapid induction (hours)
Short-lived
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Antigenic peptides
Slow induction (days)
Memory
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Sample & Assay Technologies

3. Phases of the innate immune response
Recognition
Recruitment/Activation
Effector
Recognize microbial
components
Macrophage recognition of
pathogens
inflammation
Phagocytosis and microbial
killing by PMN and M∅
Germline-encoded receptors
Inflammation leads to PMN,
monocyte (M∅/DC), NK,
eosinophil recruitment via
chemokines
Cytotoxicity by NK cells
Non-clonal (ie, all cells of the
same lineage have same
receptors)
Induction of adaptive
immunity by DC and M∅
Non-cytokines or chemokines
(complement fragments, fMLP)
recruit and activate innate
immune cells
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4. Epithelia, peptides, innate-like lymphocytes
Microbial invaders enter the body through skin and mucosa, and are met by several innate
defenses before encountering circulating cellular effectors.
.
Mucus/cilia (sweep out microbes before they can adhere)
Chemical defenses
β-Defensins (epithelial cells and leukocytes, skin, tongue, respiratory tract)
α-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 γ/δ T lymphocytes – GI
B-1 B cells – peritoneal and pleural cavities
NK-T cells – thymus, peripheral lymphoid organs
.
.
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5. Induction of innate immunity by PRRs
Pathogen-associated molecular patterns (PAMPs)
.
Lipopolysaccharide
Flagellin
Single- and double-stranded nucleic acids
CpG
Lipoteichoic acid
Zymosan
etc…
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)
http://sabiosciences.com/pathway.php?sn=Toll_Like_Receptors
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6. Cellular and molecular effectors of innate immunity
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7. Cellular components – phagocytes and natural killer cells
Macrophages
Long-lived phagocytes
Short-lived phagocytes
Mannose receptor, scavenger
receptors, CD14
Complement receptors
Granules (acid hydrolase,
myeloperoxidase, defensins,
cathepsin G, lysozyme,
lactoferrin, elastase, etc)
ROS, RNI
Respiratory burst
Proinflammatory cytokine production
(IL-1β, IL-12, TNF-α, IL-6,CXCL8)
NETs (neutrophil extracellular
traps: chromatin + serine
proteases)
Produce growth factors for tissue
remodeling
Arise from circulating monocytes
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NK cells
Neutrophils
Proinflammatory cytokines
(IL-12, TNF-α)
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Expansion/activation in response to
IL-15, IL-12, Type I IFNs
Produce IFN-γ, IL-1, and IL-2
Recognize Class I MHC (inhibitory &
activating receptors)
Cytotoxic cells (via perforin and
granzymes, or ADCC)
Blood, spleen localization
Memory?
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8. Cellular components – parasite defense
Basophils
Eosinophils
Helminths, viruses and
bacteria (also allergy)
Least abundant
granulocyte
Respond to
complement, IgE, TLR
stimulation
Histamine,
proteoglycans, lipid
mediators, IL-4, IL17E
Produce major
basic protein,
eicosanoids,
histamine,
peroxidases,
acid
phosphatase,
growth factors,
and cytokines
Mast cells
Release histamine,
heparin, proteases,
TNF and other
proinflammatory
cytokines, eicosanoids,
etc.
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Macroparasite and
helminth defense,
allergy
Helminths, viral
infection, allergy
IgE-activated,
matures via IL-3
Nuocytes
Recently identified in
Type 2 innate
response against
helminth infection
(Neill et al, Nature
2010)
Respond to IL-23 and
IL-33, produce IL-13
Involved in allergic
lung inflammation
Activated by IL5, IL-3, and GMCSF
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9. Cellular components – dendritic cells
“Professional antigen presenting cells (APCs)”, linking 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 costimulation 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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10. Complement cascade
Three types:
.
Classical
Alternative
Lectin-induced
Initiation:
.
C1q, mannose-binding lectin, or C3b bind
microbe surfaces (or C1q binds C-reactive
protein)
3 pathways converge with generation of C3
convertase through triggered-enzyme
cascades
Outcomes:
.
Opsonization
Chemotaxis/activation of leukocytes (C3a,
C5a)
Membrane-attack complex
Results from C3b generation
Consists of C5b-9
Effective against Neisseria species
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11. Cytokines and Chemokines
Cytokines produced by innate immune cells
Type I IFNs, IFN-γ
TNF-α
IL-1, 6, 8, 10, 12, 15, 18
.
Cytokines that enhance/inhibit innate immune activity
Enhance: IFN-γ, 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, fibroblasts,
recruits monocytes, DCs, NK cells)
.
.
http://sabiosciences.com/pathway.php?sn=Cytokine_Network
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12. Important signaling networks
Cytokines
JAK/STAT
MAPK
IRFs
PI3K
NFkappaB
.
Chemokines
PI3K/AKT
MAPK
NFkappaB
.
TLRs
.
NFkappaB
JNK/MAPK
IRFs
PI3K
http://sabiosciences.com/pathway.php?sn=Chemokine_Signaling
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13. 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 antigen to T cells and provide costimulation
Chemokine and cytokine production
lymphocyte expansion, activation
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14. Technologies for innate immune research
Pathway-focused gene expression analysis
Featured publications: Derbigny, W.A. et al. 2012,
Infect. Immun.
RT2 Profiler PCR Array, Mouse Inflammatory
Cytokines
Signaling pathway reporter arrays
Featured publication: Zughaier, S.M. 2011, J.
Leukocyte Biol.
Cignal Finder 10-Pathway Reporter Array
RT2 Profiler PCR Array, Human TLR and Human
Apoptosis
Cytokine analysis
Featured publication: Rahman, S. et al. 2011 J.
Immunol.
Multi-Analyte ELISArray
RT2 Profiler PCR Array, Mouse IFN-α and IFN-β
Response, Mouse Dendritic and Antigen
Presenting Cells
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15. RT2 Profiler PCR Arrays
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-α/β Response
NFkB Signaling
MAPK Signaling
PI3K/AKT Signaling
(and more… 140+ pathways,
including custom arrays)
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16. Application data – which cytokines alter expression
after PMA-Ionomycin treatment?
Human PBMCs were treated with PMA and ionomycin, and then analyzed using the Common
Cytokines RT2 Profiler PCR Array. This volcano plot shows both fold-change and the statistical
significance, and demonstrates that 23 genes, including IL-10, IFN-gamma, IL-2, and TNF
were upregulated, while IL-1beta and 5 other genes were downregulated in response to
treatment.
.
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17. How does gene expression change during infection?
Characterizing overall responses of gene networks can give 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?
.
Identifying a role for TLR3 in the innate immune response to
.
Chlamydia muridarum infection in murine oviduct epithelial cells
Derbigny, W.A. et al. 2012, Infect. Immun.
.
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-β in initiating inflammatory
responses against an intracellular bacterial pathogen.
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18. Application data – TLR3 in the innate immune response
Compared gene expression changes under the following conditions using the Mouse
Inflammatory Cytokines PCR Array:
Wild-type cells plus C. muridarum
TLR3-deficient cells plus C. muridarum alone
TLR3-deficient cells plus IFN-β and C. muridarum
TLR3-deficient cells plus IFN-β alone
.
Results
Wild-type infected cells: most chemokines and interleukins, CXCL15, CCR10.
Deleting TLR3: diminished response to infection, except CXCL15 effects. CCR9 and
Lta showed modest compared to WT cells.
IFN-β + infection of TLR3-/-: partially rescued chemokine and Casp1 responses
IFN-β sans C. muridarum: still stimulated some chemokine response, but not as
much as with infection
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19. Application data – TLR3 in the innate immune response
Conclusion
TLR3 and IFN-β 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 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.
RT2 Profiler PCR Arrays provide an excellent tool for identifying which genes in a
specific biological pathway are affected by infection, cytokine stimulation, and gene
knockdown.
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20. Technologies for innate immune research
Pathway-focused gene expression analysis
Featured publications: Derbigny, W.A. et al. 2012,
Infect. Immun.
RT2 Profiler PCR Array, Mouse Inflammatory
Cytokines
Signaling pathway reporter arrays
Featured publication: Zughaier, S.M. 2011, J.
Leukocyte Biol.
Cignal Finder 10-Pathway Reporter Array
RT2 Profiler PCR Array, Human TLR and Human
Apoptosis
Cytokine analysis
Featured publication: Rahman, S. et al. 2011 J.
Immunol.
Multi-Analyte ELISArray
RT2 Profiler PCR Array, Mouse IFN-α and IFN-β
Response, Mouse Dendritic and Antigen
Presenting Cells
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21. Cignal Reporter Assays & Arrays
Functionally verified assays for 45 pathways:
Type I IFN
IFN-γ
NFκB
MAPK
PI3K/AKT
STAT3
TGF-β
And more…
.
Cignal Finder 10-Pathway Arrays:
Cancer
Immune Signaling
Development
Stem Cell & Differentiation
Nuclear Receptors
Stress & Toxicity
.
Cignal 45-Pathway Array
.
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22. 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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23. 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?
.
Neisseria meningitidis capsular polysaccharides induce
inflammatory responses via TLR2 and TLR4-MD-2
Zughaier, S. M., J. Leukoc. Biol., March 2011
.
.
Context:
Research aim: determining how CPS-triggered signaling proceeds
Human TLR Signaling and Human Apoptosis RT2 Profiler PCR Arrays
Cignal Finder 10-Pathway Reporter Array to identify active signaling pathways
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24. Application: Reporter assay in immune response
Pathways interrogated:
.
NFκB
PKC/Ca++ (NFAT)
Type I IFNs (ISRE)
IFN-γ (GAS)
MAPK/ERK (SRE)
MAPK/JNK (AP-1)
TGF-β (SMAD)
cAMP-PKA (CRE)
C-EBP
Glucocorticoid receptor (GRE)
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-MD2 were identified as the CPS receptors on macrophages.
TLRs 2 and 4 can signal through NFκB or MAPK pathways. Which ones for CPS?
Purified CPS from LOS-deficient strain of N. meningitidis (IpxA mutant)
Dosed HEK/TLR2/6 or HEK/TLR4-MD2-CD14 cells 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
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25. Application: Reporter assay in immune response
.
Findings:
Strong NFκB reporter activity in TLR4-MD2 and TLR2-6 stably transfected cells
stimulated with serogroup B CPS
Milder activity from Type I IFN/ISRE (TLR2-6), MAPK-JNK/AP-1 (both), and MAPERK/SRE (TLR4-MD2).
Also observed variation in gene expression profiles between cells stimulated with CPSlpxA vs LOS (including TNF, NOD1, CD40LG, LTA, and CARD6).
.
Conclusions:
Cignal Finder 10-Pathway Reporter Array identified 4 inflammatory signal transduction
pathways potentially involved in TLR-mediated recognition of CPS
The RT2 Profiler PCR Array determined that CPS induces qualitatively distinct gene
expression responses compared to LOS
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Sample & Assay Technologies

26. Technologies for innate immune research
Pathway-focused gene expression analysis
Featured publications: Derbigny, W.A. et al. 2012,
Infect. Immun.
RT2 Profiler PCR Array, Mouse Inflammatory
Cytokines
Signaling pathway reporter arrays
Featured publication: Zughaier, S.M. 2011, J.
Leukocyte Biol.
Cignal Finder 10-Pathway Reporter Array
RT2 Profiler PCR Array, Human TLR and Human
Apoptosis
Cytokine analysis
Featured publication: Rahman, S. et al. 2011 J.
Immunol.
Multi-Analyte ELISArray
RT2 Profiler PCR Array, Mouse IFN-α and IFN-β
Response, Mouse Dendritic and Antigen
Presenting Cells
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Sample & Assay Technologies

27. Multi-Analyte ELISArrays
Common Cytokines
Inflammatory Cytokines
TLR-induced Cytokines I: Viral
TLR-induced Cytokines II: Microbial
Autoimmunity
Th1 / Th2 / Th17 Cytokines
Common Chemokines
.
.
.
.
.
.
.
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28. Application data – are Th1 or Th2
cytokines being produced?
Time-dependent (0, 6, 18, 24, and 48 h) patterns of Th1/Th2 cytokine induction by human
peripheral blood mononuclear cells (PBMC) in response to PMA (50 µg/ml) and ionomycin (1
µg/ml) were monitored. The relative amount of each cytokine was profiled at the same time using
the ELISArray Kit.
.
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29. What comprises the cytokine milieu?
Innate immune responses produce a multitude of cytokines, which play distinct
roles. Is it possible to efficiently detect several related cytokines simultaneously?
.
Murine FLT3 Ligand-Derived Dendritic Cell-Mediated Early Immune
.
Responses Are Critical to Controlling Cell-Free Human T Cell Leukemia Virus
Type 1 Infection
Rahman, S. et al. J. Immunol. 2011
.
Context:
.
Research aim: to clarify how DCs respond to HTLV-1 infection
RT2 Profiler PCR Arrays for IFNα/β 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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30. Case study, Rahman et al.
Cytokines detected:
.
IL-2
IL-4
IL-5
IL-6
IL-10
IL-12
IL-13
IL-17A
IL-23
IFN-γ
TNF-α
TGF-β1
Study background
.
Innate immunity was not thought to have a major part in HTLV-1 control; however,
the authors demonstrate a significant role for Flt3L-generated DC and Type I IFN in
control of cell-free virus (the type that would be encountered early in infection)
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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Sample & Assay Technologies

31. Case study, Rahman et al.
Findings
.
Multi-Analyte ELISArray – proinflammatory cytokines were secreted by DCs after
viral challenge (IL-6, TNF-α, IL-12). Th2 cytokines (IL-4, IL-5, IL-13) were not
induced, but IL-10 and TGF-β were secreted as well.
RT2 Profiler PCR Arrays
IFNα, IFNβ 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/presentation genes were also enhanced. Many key chemokines (CCL2,
7, 8) and receptors (CCR5, CCR3) were downregulated.
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 PCR Arrays 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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32. Summary
The innate immune system comprises 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 genes simultaneously, and are
available for over 140 pathways. Many of these are related to innate immunity and
host defense, and 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 analysis assays using a traditional ELISA format.
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Sample & Assay Technologies

33. Thank You for Attending!
If you are interested in trying out these technologies in your research, please
visit:
.
http://www.sabiosciences.com/rt_pcr_product.php
.
http://www.sabiosciences.com/reporterassays.php
.
http://www.sabiosciences.com/ELISA.php
.
If you have any questions about using our products in your research, please
contact us at support@sabiosciences.com.
.
Thank you for your time and attention!
.
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