innate immunity 2013

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

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Innate and adaptive immunity

� PAMPs/DAMPs� Rapid induction (hours)� Short-lived

� Antigenic peptides� Slow induction (days)� Memory

InnateAdaptive


Phases of the innate immune response

Recognition

Recognize microbial components

Germline-encoded receptors

Non-clonal (ie, all cells of the same lineage have same receptors)

Recruitment/Activation

Macrophage recognition of pathogens � inflammation

Inflammation leads to PMN, monocyte (M∅/DC), NK, eosinophil recruitment via chemokines

Non-cytokines or chemokines(complement fragments, fMLP) recruit and activate innate immune cells

Effector

Phagocytosis and microbial killing by PMN and M∅

Cytotoxicity by NK cells

Induction of adaptive immunity by DC and M∅


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


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


Cellular and molecular effectors of innate immunity


Cellular components – phagocytes and natural killer cells

Neutrophils NK cells

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?

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-α)

Macrophages

Long-lived phagocytes

Mannose receptor, scavenger receptors, CD14

Complement receptors

ROS, RNI

Proinflammatory cytokine production (IL-1β, IL-12, TNF-α, IL-6,CXCL8)

Produce growth factors for tissue remodeling

Arise from circulating monocytes


Cellular components – parasite defense

Mast cells Basophils

Produce major basic protein, eicosanoids, histamine, peroxidases, acid phosphatase, growth factors, and cytokines

Helminths, viral infection, allergy

Activated by IL-5, IL-3, and GM-CSF

Least abundant granulocyte

Histamine, proteoglycans, lipid mediators, IL-4, IL-17E

Macroparasite and helminth defense, allergy

IgE-activated, matures via IL-3

Eosinophils

Helminths, viruses and bacteria (also allergy)

Respond to complement, IgE, TLR stimulation

Release histamine, heparin, proteases, TNF and other proinflammatory cytokines, eicosanoids, etc.

Nuocytes

Recently identified in Type 2 innate response against helminth infection (Neill et al, Nature2010)

Respond to IL-23 and IL-33, produce IL-13

Involved in allergic lung inflammation


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


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


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


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


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


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


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 ResponseInflammatory Cytokines & ReceptorsDendritic & Antigen-Presenting CellsInflammasomesIFN-α/β ResponseNFkB Signaling MAPK Signaling PI3K/AKT Signaling

(and more… 140+ pathways, including custom arrays)


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.


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 re sponse 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.


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


Conclusion

� TLR3 and IFN-β are major mediators of the inflammatory response to C. muridarumin 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.

Application data – TLR3 in the innate immune response





Cytokines



Apoptosis





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


Application data: determining the signaling pathway s 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.


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


Application: Reporter assay in immune response

.Pathways interrogated: � NFκB MAPK/JNK (AP-1)� PKC/Ca++ (NFAT) TGF-β (SMAD)� Type I IFNs (ISRE) cAMP-PKA (CRE)� IFN-γ (GAS) C-EBP� MAPK/ERK (SRE) 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


.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 MAP-ERK/SRE (TLR4-MD2).

� Also observed variation in gene expression profiles between cells stimulated with CPS-lpxA 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

Application: Reporter assay in immune response





Cytokines



Apoptosis





Multi-Analyte ELISArrays

.Common Cytokines

.Inflammatory Cytokines

.TLR-induced Cytokines I: Viral

.TLR-induced Cytokines II: Microbial

.Autoimmunity

.Th1 / Th2 / Th17 Cytokines

.Common Chemokines


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.


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 Hum an 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


Case study, Rahman et al.

.Cytokines detected: � IL-2 IL-13� IL-4 IL-17A� IL-5 IL-23� IL-6 IFN-γ� IL-10 TNF-α� IL-12 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 infect ion? � 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.


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.


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.


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 [email protected].

.Thank you for your time and attention!

innate immunity 2013

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