Download Host-pathogen interactions_Oct 2015

Welcome to our four-part webinar series on microbiology
The Microscopic War: Microbes and Immunity
A 4-Part Webinar Series
Part 1: Host–Pathogen Interactions: Molecular Basis and Host Defense
Mechanisms
Part 2: Microbiome: From Identification to Characterization
Part 3: Exploring the First Line of Defense: Research Tools for the Innate
Immune System
Part 4: Toll-like Receptors in Inflammation
Sample to Insight
Host – pathogen interactions
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Host - Pathogen Interactions: Molecular Basis and Host Defense Mechanisms
Contact Technical Support:
[email protected]
1-800-362-7737
Webinar-related questions:
[email protected]
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Host – pathogen interactions
2
Welcome to our four-part webinar series on microbiology
The Microscopic War: Microbes and Immunity
A 4-Part Webinar Series
Part 1: Host–Pathogen Interactions: Molecular Basis and Host Defense
Mechanisms
Part 2: Microbiome: From Identification to Characterization
Part 3: Exploring the First Line of Defense: Research Tools for the Innate
Immune System
Part 4: Toll-like Receptors in Inflammation
Sample to Insight
Host – pathogen interactions
3
Legal disclaimer
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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Host – pathogen interactions
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Agenda
1
Introduction to the microbiome
2
The host-pathogen interaction
3
The host defense mechanisms
4
Technologies for exploring host-pathogen
interactions
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Host – pathogen interactions
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Introduction to the microbiome
Cellular composition of the organism
Human
Estimations of the number of microbial cells that live in and
on the human body, human cells are outnumbered by a factor
of 10.
35 trillion human cells = 350 trillion microbial cells!
What does “microbiome” mean?
Microbes are microscopic organisms that can be either
single or multicellular.
Microbiota Microbiota are the microbes that live in a specific location,
e.g. the human body, the gut, skin, soil, etc.
Human microbiome is defined as the collective
genomes of the complete microbiota present in a human
body.
Metagenomics is the study of the collection of genomes
derived from a specific sample or community.
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NIH Human Microbiome Project (HMP)
The Common Fund's (HMP) is developing research resources to enable the
study of the microbial communities that live in and on our bodies and the
roles they play in human health and disease.
The first phase of HMP (FY2007-2012)
Characterize the composition and diversity of microbial
communities which inhabit major mucosal surfaces of the
human body, and evaluated the genetic metabolic
potential of these communities.
~10,000 organisms live with us (10 times more)
~ 8 ×106 genes in this genome (20-60% are not cultivable)
The second phase of HMP (FY2013-2015)
Develop tools and datasets for the research community for
studying the role of these microbes in human health and
disease.
The HMP plans to sequence 3000 genomes from both cultured and uncultured bacteria, plus several
viral and small eukaryotic microbes isolated from human body site.
http://hmpdacc.org/
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Microbiome is everywhere
The Microbiome: the trillions of microorganisms maintain health and cause disease in humans
SnapShot: The Human Microbiome
Different body sites have unique
communities
The gut microbiota is profoundly altered during
pregnancy
Microbial diversity is changed during pregnancy
Each area of the body has its own
Shift in bacterial diversity is unrelated to health state
microbiome
Newborn babies are born with a distinct gut microbiome
The human microbiome plays a roles in many human diseases: diabetes, rheumatoid arthritis,
muscular dystrophy, multiple sclerosis, fibromyalgia, CNS related, such as schizophrenia, depression,
bipolar disorder, and some cancers.
Hoffmann A.R., et al. “The Microbiome: The Trillions of Microorganisms That Maintain Health and Cause Disease in Humans and Companion Animals.” Vet Pathol. 2015
Koren O. et al. “Host Remodeling of the Gut Microbiome and Metabolic Changes during Pregnancy”, Cell, 2012, 150, 470
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Agenda
1
Introduction to the microbiome
2
The host-pathogen interaction
3
The host defense mechanisms
4
Technologies for exploring host-pathogen
interactions
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Host – Microbiota Interaction
Dual necessity: peacefully coexist to achieve a mutually beneficial
relationship
The host provides the microbiota a niche with a stable nutrient
supply
The microbiota performs essential functions for host physiology,
including metabolic, digestive, and immune mechanisms
Regulate the host’s metabolic function and energy balance
Provide the host with the capacity to hydrolyze complex plant sugars,
synthesize vitamins, and detoxify xenobiotics in a mutualistic context
Affect the most fundamental of host physiological phenotypes, the
rate of aging itself.
Commensal gut microbiota protects the host from infection via both
direct and indirect mechanisms
The symbiosis between microbes and humans provides a stable and
common metabolic pattern and well-balanced physiological
homeostasis
Caroline Heintz and William Mair, “You Are What You Host: Microbiome Modulation of the Aging Process”, 2014, Cell, 156:408
Kamada N, et al. “Role of the gut microbiota in immunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
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Host – Microbiota Interaction
Direct and indirect protection mechanisms of microbiota
Kamada N, et al. “Role of the gut microbiota in immunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
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Host – Microbiota Interaction
Direct and indirect protection mechanisms of microbiota
Kamada N, et al. “Role of the gut microbiota in immunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
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Host – Microbiota Interaction
Direct and indirect protection mechanisms of microbiota
Kamada N, et al. “Role of the gut microbiota in immunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
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Host – Microbiota Interaction
The big question? How, what, who in the microbiota does what, when and how to
the immune system?
Identify microbiota
Virulence factors
Antibiotic resistance genes
Kamada N, et al. “Role of the gut microbiota in immunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
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The crosstalk between the microbiome and the immune system
How does microbiota shape immunity - the cellular and molecular mediators
Impact lymphoid structure development and
epithelial function
Enhance innate immunity to pathogens
Shape T cell subsets
Provide protective roles against systemic
infection
Influence invariant T cells and innate lymphoid
cells
Trigger inflammation in immunocompromised
hosts
Protect against autoimmune diseases
Lora V. Hooper et al., Interactions Between the Microbiota and the Immune System. 2012, Science 336:1268
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The crosstalk between the microbiome and the immune system
The control of immune system over microbiota
“The mammalian immune system plays an essential
role in maintaining homeostasis with resident
microbial communities, thus ensuring that the
mutualistic nature of the host-microbial relationship is
maintained.”
The critical controls of the immune system
1.
Exerts control over stratification and
compartmentalization of the microbiota
2.
Exerts control over microbiota composition
The immune system exerts critical controls over
microbiota composition, diversity, and location.
Lora V. Hooper et al., Interactions Between the Microbiota and the Immune System. 2012, Science 336:1268
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The importance of microbiota in human health and diseases
When the mutualistic relationship between the host and microbiota is disrupted, the gut
microbiota can cause or contribute to diseases
Diseases influenced by gut microbial metabolism
Kinross et al. “Gut microbiome-host interactions in health and disease.” 2011, Genome Medicine, 3:14. Review.
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The host responses
The challenge that the host faces
It is important to note that commensal bacteria do not
always protect against pathogenic infection and in certain
contexts they can facilitate it.
And under certain conditions, particular bacterial
populations can acquire pathogenic properties.
How does the host discriminate between symbiotic and
pathogenic bacteria to adjust its level of immune
response?
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Agenda
1
Introduction to the microbiome
2
The host-pathogen interaction
3
The host defense mechanisms
4
Technologies for exploring host-pathogen
interactions
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The immune responses – innate and adaptive
Innate immunity and adaptive immunity
The innate immune system is the first line of
defense against pathogens and is initiated by
genome-encoded pattern recognition receptors
(PRRs) that recognize PAMP
Non-specific and does not confer long-lasting
immunity (memory)
Immune cells: dendritic cells (DCs) and
macrophages, intestinal epithelial cells and
myofibroblasts
The adaptive immunity is highly specific,
confers long lasting immunity, and adaptable
Cooperate with the molecules and cells of the innate
immune system to mount an effective immune
response
Key players: T cells – Th1, Th2 or Th17 cell
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Innate immune responses – Toll-like receptors
Pattern recognition receptors (PRRs) include Toll-like
receptors (TLRs), NOD-like receptors (NLRs) and RIG1like receptors (RLRs), etc.
•
PRRs signaling cascades result in nuclear factor (NF)-kB
activation of gene transcription and production of proinflammatory mediators
•
PRRs also play a crucial role in the crosstalk between innate
and adaptive immune responses by promoting antigen
presenting cell maturation and T cell activation
•
TLRs induces the expression of genes required for the
inflammatory response, including inflammatory cytokines,
chemokines, antimicrobial molecules, and major
histocompatibility (MHC) and costimulatory molecules
important for adaptive immune activation.
Download map:
https://www.qiagen.com/pathway-details.aspx?pwid=445
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Adaptive immune responses
Th1 and Th2 cells
Th17 cells
Regulatory T cells
During active inflammation, Th0 cells differentiate
into T helper cells Th1, Th17 and Th2
Adaptive responses in the gut
Th1 cells produce interferon (IFN)-γ and
tumor necrosis factor (TNF)-α
Th2 cells are a major source of IL-13
Th17 cells release IL-17 and IL-21
Important adaptive immune cells
pDC detect viral antigen and release type I IFN
•Activation of mDC, mV, CD4 , and CD8 T cells
•FRCs secrete chemokines and T cell survival
factors
•FDCs coordinate B cell migration and B cell and
CD4 T cell interactions
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Host defense mechanisms - Summary
Lance W. Peterson and David Artis, “Intestinal epithelial cells: regulators of barrier function and immune homeostasis”, Nat Rev
Immunol. 2014, 14(3):141-53.
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Agenda
1
Introduction to the microbiome
2
The host-pathogen interaction
3
The host defense mechanisms
4
Technologies for exploring host-pathogen
interactions
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Technologies for exploring host-pathogen interactions
Characterize the composition and function of the gut
microbiome and the contribution of microbes to
human health.
Genomics – characterize DNA
Transcriptomics – characterize RNA
Metabolomics – characterize small molecules
Whether changes in the composition of the microbiome are associated
with or cause a disease?
Which microbial biochemical functions change in a disease at the DNA,
RNA, protein, and metabolite levels?
How do metabolic processes change in a disease?
How does an intervention or a treatment affect the composition and
biomolecular function of the microbial community?
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Technologies for exploring host-pathogen interactions
Human microbiome sample
Extract DNA
16S rRNA gene
sequencing
18S rRNA gene
sequencing
Extract RNA
Total DNA sequencing
(shotgun)
RNA expression profiling
(transcriptomics)
Gene content
Bacteria and
Archaea
Fungus
/Yeast
Gene
expression
Extract small molecules
Mass spectroscopy
(metabolomics)
Metabolite
characterization
Viruses
Identify relative frequencies and pathways
What organisms are present and what is
their relative abundance?
What are the functions of the community?
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The research question
How can we identify the microbiome and
monitor innate and adaptive immune
responses?
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Technologies for exploring host-pathogen interactions
QIAamp Fast DNA Stool Mini Kit
QIAamp UCP Pathogen Mini Kit
QIAamp UCP PurePaghogen Blood Kit
QIAamp MinElute Media Kit
Detect microbial species, virulence genes, or antibiotic
resistance genes
Microbial DNA qPCR
Assay/Assay
RNA expression
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Detecting microbial metagenomes – a complete solution
QIAGEN provides next-generation sequencing technologies for metagenomics, as well as qPCR assays
and arrays for verification of sequencing results and screening for specific bacterial species, virulence
factor genes, and antibiotic resistance genes.
Sample to Insight workflow
Sample
collection
• Allprotect Tissue
Reagents
DNA
purification
• QIAamp UCP Pathogen Mini
Kit
• QIAamp DNA Stool Mini Kit
• QIAamp UCP PurePathogen
Blood Kit
DNA
amplification
• REPLI-g Single Cell
Kit
Library
preparation
• GeneRead Library Prep
Kits
• GeneRead Size
Selection Kit
• GeneRead Library
Quant System
Verification
by qPCR
• Microbial DNA qPCR
Arrays
• Microbial DNA qPCR
Assays
• Custom Microbial DNA
qPCR Arrays
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QIAGEN’s Microbial DNA qPCR assay pipeline
Reveal the mysteries of the microbiome with over 500 assays that target speciesspecific or gene-specific microbial DNA
>500 bacteria identification assay
8 Fungi identification assay
1 Protist identification assay
87 Antibiotic resistance genes
87 Virulence factor genes
18 Arrays
DNA
purification
Detection
by qPCR
Data
analysis
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Antibiotic Resistance Genes
Bacterial Vaginosis
Biodefense
Food testing: Dairy
Food testing: Meat
Food testing: Poultry
Food testing: Seafood
Food testing: Vegetable
Intestinal Infections
Intestinal Infections 2
Metabolic Disorders
Oral Disease
Respiratory Infections
Respiratory viral
Sepsis
Urinary Tract Infections
Vaginal Flora
Water Analysis
Part 2 webinar: Microbiome: From Identification to Characterization
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Technologies for exploring host-pathogen interactions
A complete workflow for host responses profiling
Sample
collection
Sample
preparation
RNA
purification
Gene
expression
analysis
Data
analysis
Profiling gene expression from immune cells (especially
cytokines, chemokines and other immune molecules)
can be interpreted into immune system “status”
Inflammation?
Early
Chronic
Resolution
Type of response (bacterial, viral, other)?
RT2 Profiler PCR Arrays
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Host response profiling: gene expression
Profile 84 or 370 genes simultaneously with RT2 Profiler PCR Arrays
Antifungal Response
Antiviral Response
Antibacterial Response
Innate & Adaptive Immune Responses
Inflammatory Cytokines & Receptors
Dendritic & Antigen-Presenting Cells
Inflammasomes
Th1 & Th2 Responses
Toll-Like Receptor Signaling Pathway
IFN-a/b Response
NFkB Signaling
NFkB Signaling Targets
MAPK Signaling
PI3K/AKT Signaling
140+ pathways, including custom arrays
Wet-bench validated assays for all genes of 13 species
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PCR Arrays format and contents
Each well contains lyophilized – “Verified qPCR Assay”
84 pathway-specific genes of interest
5 housekeeping genes
Genomic DNA contamination control
(GDC)
Reverse transcription controls (RTC), n=3
Positive PCR controls (PPC), n=3
ACTB, B2M, GAPDH, HPRT1, RPL0
Customizable – Add 4 genes to a catalog
RT2 Profiler PCR Array, or Completely
customize based on your research.
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How RT2 Profiler PCR arrays work?
Isolate total RNA
cDNA Synthesis
Control
Sample
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
One sample per PCR Array
Two minutes with multi-channel pipet
Run 40 cycle qPCR Program
Standard cycling conditions for all real
time PCR instruments
2 hours
Upload and Analyze Data (FREE)
15 minutes from raw Ct to fold
change data
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How RT2 Profiler PCR arrays work?
Isolate total RNA
cDNA Synthesis
Control
Sample
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
One sample per PCR Array
Two minutes with multi-channel pipet
Run 40 cycle qPCR Program
Standard cycling conditions for all real
time PCR instruments
2 hours
Upload and Analyze Data (FREE)
15 minutes from raw Ct to fold
change data
Sample to Insight
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How RT2 Profiler PCR arrays work?
Isolate total RNA
cDNA Synthesis
Control
Sample
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
One sample per PCR Array
Two minutes with multi-channel pipet
Run 40 cycle qPCR Program
Standard cycling conditions for all real
time PCR instruments
2 hours
Upload and Analyze Data (FREE)
15 minutes from raw Ct to fold
change data
Sample to Insight
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How RT2 Profiler PCR arrays work?
Isolate total RNA
cDNA Synthesis
Control
Sample
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
One sample per PCR Array
Two minutes with multi-channel pipet
Run 40 cycle qPCR Program
Standard cycling conditions for all real
time PCR instruments
2 hours
Upload and Analyze Data (FREE)
15 minutes from raw Ct to fold
change data
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RT2 Profiler PCR Array data analysis
Free complete & easy analysis with web/excel-based software
Multiple analysis formats to interpret gene expression results
Scatter Plot
Clustergram
Volcano Plot
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Application data
How are cytokines regulated under 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.
Next step – verify results by ELISA for only the cytokines which are changing!
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Title, Location, Date
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Host response profiling: gene expression
RNA expression
Attend the part 3 &4 webinars
Part 3: Exploring the First Line of Defense: Research Tools for the Innate Immune System
Part 4: Toll-like Receptors in Inflammation – The bridge between innate and adaptive immunity
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Title, Location, Date
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Topics covered
1
Introduction to the microbiome
2
The host-pathogen interaction
3
The protection mechanisms of microbiota
The control of immune system over microbiota
The host defense mechanisms
4
Human microbiome - microbiota
The complex host-microbe relationship
The impact of microbiome on human health and disease
Innate immune response
Adaptive immune response
Technologies for exploring host-pathogen
interactions
Characterize the composition and function of the gut microbiome
Profile innate and adaptive immune response
Sample to Insight
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Welcome to our four-part webinar series on microbiology
The Microscopic War: Microbes and Immunity
A 4-Part Webinar Series
Part 1: Host–Pathogen Interactions: Molecular Basis and Host Defense
Mechanisms
Part 2: Microbiome: From Identification to Characterization
Part 3: Exploring the First Line of Defense: Research Tools for the Innate
Immune System
Part 4: Toll-like Receptors in Inflammation
Sample to Insight
Host – pathogen interactions
42
Thank you for attending
Questions?
Contact QIAGEN Technical Service
Call: 1-800-426-8157 for US
Call: +49 2103-29-12400 for EU
Email:
[email protected]
[email protected]
[email protected]
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